Materials and methods for prevention and treatment of RNA viral diseases

ABSTRACT

The subject invention concerns a method of inhibiting an RNA virus infection within a patient by increasing the amount of 2-5 oligoadenylate synthetase (2-5 AS) activity within the patient. Preferably, the preventative and therapeutic methods of the present invention involve administering a nucleotide encoding 2-5 AS, or at least one catalytically active fragment thereof, such as the p40, p69, p100 subunits, to a patient in need thereof. The present inventors have determined that overexpression of 2-5AS causes a reduction in epithelial cell damage, reduction in infiltration of mononuclear cells in the peribronchiolar and perivascular regions, and reduction in thickening of the septa in the lungs. Levels of chemokines, such as MIP1-α, are also reduced upon overexpression of 2-5AS. The subject invention also pertains to pharmaceutical compositions containing a nucleotide sequence encoding 2-5 AS and a pharmaceutically acceptable carrier, as well as vectors for delivery of the 2-5 AS nucleotide sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional patentapplication Serial No. 60/319,216, filed Apr. 30, 2002, and provisionalpatent application Serial No. 60/319,313, filed Jun. 12, 2002, which arehereby incorporated by reference in their entirety, including allnucleic acid sequences, amino acid sequences, figures, tables, anddrawings.

BACKGROUND OF INVENTION

[0002] Respiratory syncytial virus (RSV) is a major respiratory pathogenin infants, young children, and the elderly, causing severebronchiolitis, pneumonia, and exacerbation of asthma. In the UnitedStates alone, RSV causes approximately 4 million cases of respiratorytract infection annually, which results in 149,000 hospitalizations and11,000 deaths. It has been established that interferon-gamma (IFN-γ)gene therapy is effective against RSV infection in BALB/c mice (Kumar etal., Vaccine 18, 558-567, 1999).

[0003] Intranasal administration of a plasmid expressing IFN-γ cDNAproved to be an effective prophylaxis in mice. Furthermore, IFN-γexpressed by a recombinant respiratory syncytial virus attenuates virusreplication in mice without compromising immunogenicity. IFN-γ, a typeII interferon, is a pleotropic cytokine which plays an important role inmodulating nearly all phases of immune and inflammatory responses. IFNsbind to specific receptors on cells and activate the JAK-STAT signalingcascade, which culminates in the transcriptional induction ofIFN-stimulated genes (ISGs). The Jak1 and Jak2 phosphorylate STAT-1following the binding of IFN-γ to its receptor. Once phosphorylated,STAT molecules dimerize and translocate to the nucleus and bind to gammaactivated sequence (GAS) elements present in the regulatory regions ofvarious ISGs. The antiviral mechanism of IFN-γ may involve one or moreof a number of ISG-encoded products, including interferon regulatoryfactor-1 (IRF-1) double stranded RNA-dependent protein kinase (PKR), theMx family of proteins, a family of 2′-5′-oligoadenylate synthetases (2-5AS), and RNase L.

[0004] RNase L is constitutively expressed in most mammalian cells andis found in an inactive form bound to RNase L inhibitor (RLI), a 68 kDaprotein not regulated by IFN-γ. The 2-5 AS produces a series of 5′phosphorylated, 2′, and 5′-linked oligoadenylates (2-5A) from ATP, whenactivated by double-stranded ribonucleic acid (dsRNA). Upon binding of2-5 AS with RNase L, RLI is released and consequently, RNase L isdimerized and activated, mediating the cleavage of single-stranded RNA.However, the mechanism of the induction and activation of each of thesegenes is different in different cells and for the types of viruses. Themechanism of the IFN-γ-mediated anti-viral activity remains to beelucidated for many clinically important viruses.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides materials and methods useful forinhibiting viral infections caused by ribonucleic acid (RNA) virusesthat transiently produce double-stranded RNA during replication. Thesubject invention concerns therapeutic methods for preventing ordecreasing the severity of symptoms associated with an RNA viralinfection by increasing endogenous levels of 2′-5′ oligoadenylatesynthetase (2-5 AS) activity within the patient. Preferably, theendogenous levels of the 2-5 AS p40 subunit (e.g., the 40 kDa, 42 kDa,46 kDa, or other splice variants), p69 subunit, (e.g., the 69 kDa, 71kDa, or other splice variants), p100 subunit, or combinations thereof,are increased within the patient.

[0006] The materials and methods of present invention are effective fortreating or preventing human or animal infections from RNA viruses suchas, members of the family paramyxoviridae, respiratory syncytial virus(RSV), Rhinovirus, Vaccinia, Reovirus, human immunodeficiency virus(HIV), encephalomyocarditis virus (EMCV), Hepatitis B, Hepatitis C, aswell as bovine respiratory syncytial virus (BRSV), which infect cattle,sheep, and goats; Measles virus; Sendai virus; Parainfluenza 1, 2, and3; Mumps virus, Simian virus; and Newcastle virus.

[0007] In one aspect, the method of the present invention involves theadministration of a nucleotide sequence encoding 2-5 AS, or at least onecatalytically active fragment of 2-5 AS, such as the p40, p69, or p100subunits of 2-5 AS, to a patient in need thereof. The nucleotidesequence encoding 2-5 AS or at least one catalytically active fragmentthereof can be administered to the patient, for example, in a viralvector or non-viral vector, such as plasmid deoxyribonucleic acid (DNA).In cases wherein the RNA virus is one which infects the patient'srespiratory system, the nucleotide sequence encoding 2-5 AS, or at leastone catalytically active fragment thereof, is preferably administeredorally or intranasally to the epithelial mucosa of the respiratorysystem.

[0008] The present invention also pertains to pharmaceuticalcompositions comprising a nucleotide sequence encoding 2-5 AS, or atleast one catalytically active fragment thereof, such as the p40, p69,or p100 subunits of 2-5 AS, and a pharmaceutically acceptable carrier.The pharmaceutical compositions of the present invention are useful forpreventing or decreasing the severity of symptoms associated with RNAviral infections. In another embodiment, the pharmaceutical compositionsof the present invention comprise the 2-5 AS polypeptide, or at leastone catalytically active fragment of the 2-5 AS polypeptide, and apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe present invention can include various agents that protect thenucleic acid or amino acid contents from protein degradation.

[0009] In another aspect, the present invention concerns vectorscontaining a nucleotide sequence encoding 2-5 AS, or at least onecatalytically active fragment thereof, such as the p40, p69, or p100subunits of 2-5 AS. Optionally, the vector can further include apromoter sequence operatively linked to the nucleotide encoding 2-5 ASor at least one catalytically active fragment thereof, permittingexpression of the nucleotide sequence within a host cell. In anotheraspect, the present invention includes host cells that have beengenetically modified with a nucleotide sequence encoding 2-5 AS, or atleast one catalytically active fragment thereof, such as the p40, p69,or p110 subunits of 2-5 AS.

BRIEF DESCRIPTION OF DRAWINGS

[0010] FIGS. 1A-D show the results of pre-incubation of HEp-2 cells for4-20 hours with different concentrations of IFN-γ and subsequentinfection with RSV.

[0011]FIGS. 2A and 2B show results of a western blot analysis usingspecific antibodies to IRF-1, IRF-2, cytokeratin-18, double stranded RNAprotein kinase (PKR), and inducible nitric oxide synthase (iNOS).Proteins were analyzed from cells at various time points post treatmentwith IFN-γ (1000 U/ml). Cytokeratin-18 was used as an internal control.

[0012]FIG. 3 show results of northern analysis performed using genespecific probes for IRF-1 and the p40 and p69 isoforms of 2-5 AS.

[0013]FIG. 4 shows results of exposure of HEp-2 cells to IFN-γ (1000U/ml at 20 hours pre-infection) and treatment with equimolar mixtures ofantisense oligonucleotides (ODNs) to both p40 and p69 isoforms of 2-5AS. Scrambled mismatch of the antisense ODN sequence to p40 and p69 atthe same concentration were used as control.

[0014] FIGS. 5A-D show the results of northern analyses of (i) RNAs fromRNAse L inhibitor (RLI) and HEp-2 using a gene specific probe for RLIand (ii) the level of mRNA expression of IRF-1, p40, and p69 isoforms of2-5 AS.

[0015]FIG. 6 show the results of treatment of both HEp-2 cells andRLI-14 cells with IFN-γ (at 100-1000 U/ml at 20 hours pre-infection) andsubsequent infection with RSV.

[0016]FIG. 7 shows the results of treatment of HEp-2 cells with IFN-γ(at 100-1000 U/ml at 20 hours pre-infection) and subsequent infectionwith RSV. 2-5A was transfected at 3 hours prior to RSV infection. Cellswere harvested at 72 hours post infection and the clear cell homogenatewas used for the RSV plaque assay (***: p<0.005; ††: p<0.05).

[0017] FIGS. 8A-8C show lung titers of RSV in infected mice following2-5AS cDNA vaccination. BALB/c mice (n=4) were intranasally administeredwith p2′-5′ AS (25 mg of DNA each time complexed with lipofectamine) oran equal amount of empty pVAX (CLONTECH, Palo Alto, Calif., USA) vectorDNA (as a transfection control) 3 times in 2 day intervals. As shown inFIG. 8A, 2-5AS cDNA vaccination significantly attenuated lung titers ofRSV. FIG. 8B shows that vaccination with 2-5 AS cDNA decreasesproduction of the chemokine macrophage inflammatory protein-1 alpha(MIP-1 a). The results of bronchoalveolar lavage (BAL) cell differential(FIG. 8C) show that 2-5 AS does not significantly alter the cellularcomposition of the lung, although the percent of neutrophils isincreased in the lungs of mice treated with 2-5 AS cDNA.

[0018] FIGS. 9A-9C show representative photomicrographs of lungs stainedwith hematoxylin and eosin (H & E). FIG. 8A is an untreated control.FIGS. 9B and 9C show histological sections of RSV infected lungsfollowing treatment with the empty pVAX vector and p2′-5′ AS,respectively.

[0019]FIG. 10 shows results of treatment with adenoviral vector(Ad)-2-5AS (p40) results in attenuation of RSV replication. BALB/c micewere intranasally administered with Ad-p40 and then infected with RSV.Lungs were harvested five days post RSV infection and RSV replicationwas assayed by RT-PCR analysis of RSV-N gene. GAPDH was used as internalcontrol.

[0020]FIG. 11 shows that Ad-p40 attenuates RSV lung titers. Mice wereintranasally given Ad-p40 and then infected with RSV. Lungs wereharvested five days post RSV infection and lung homogenate was used forRSV plaque assay. Ad-LacZ was used as control.

[0021]FIG. 12 shows that Ad-p40 inhibits RSV induced airway reactivity.BALB/c mice were intranasally administered with Ad-p40 and subsequentlyinfected with RSV. AHR was measured on day 4 post-RSV infection. Ad-p40treatment significantly decreased pulmonary inflammation.

[0022] FIGS. 13A-13H show that Ad-p40 overexpression normalizesmacrophage and lymphocyte numbers in the lung in RSV infected mice. BALcell differential was performed and percentages of macrophage,lymphocytes and neutrophils was determined. Both Ad-IFNg and Ad-p40treatment reduced the lymphocyte population to normal, compared toRSV-infected mice. Histological sections from lungs were stained withhematoxylin and eosin and representative photomicrographs are shown.Sections shown are as follows: Naive mice (FIG. 13A; with magnifiedinset FIG. 13B); RSV infected mice (FIG. 13C; with magnified inset, FIG.13D); Ad-p40 treated mice (FIG. 13E; with magnified inset, FIG. 13F);and Ad-lacZ treated mice (FIG. 13G; with magnified inset, FIG. 13H).

BRIEF DESCRIPTION OF SEQUENCES

[0023] SEQ ID NO: 1 is a nucleotide coding sequence (CDS) for the human40 kDa splice variant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′oligoadenylate synthetase (National Center for Biotechnology Information(NCBI) Accession Number NM_(—)016816).

[0024] SEQ ID NO: 2 is an amino acid sequence of the human 40 kDa splicevariant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)016816).

[0025] SEQ ID NO: 3 is a nucleotide coding sequence (CDS) for the human46 kDA splice variant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′oligoadenylate synthetase (National Center for Biotechnology Information(NCBI) Accession Number NM_(—)016816).

[0026] SEQ ID NO: 4 is an amino acid sequence of the human 46 kDA splicevariant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)016816).

[0027] SEQ ID NO: 5 is a nucleotide coding sequence (CDS) for the human69 kDA splice variant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′oligoadenylate synthetase (NCBI Accession Number NM_(—)002535).

[0028] SEQ ID NO: 6 is an amino acid sequence of the human 69 kDa splicevariant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)002535).

[0029] SEQ ID NO: 7 is a nucleotide coding sequence (CDS) for the human71 kDA splice variant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′oligoadenylate synthetase (NCBI Accession Number NM_(—)002535).

[0030] SEQ ID NO: 8 is an amino acid sequence of the human 71 kDa splicevariant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)002535).

[0031] SEQ ID NO: 9 is a nucleotide coding sequence (CDS) for the human100 kDa subunit (“p100 subunit”) of 2′-5‘oligoadenylate synthetase’(NCBI Accession Number AF063613).

[0032] SEQ ID NO: 10 is an amino acid sequence of the human 100 kDasubunit (“p100 subunit”) of 2′-5′ oligoadenylate synthetase (NCBIAccession Number AF063613).

[0033] SEQ ID NO: 11 is a nucleotide coding sequence (CDS) for the mousehomolog of the 2′-5′ oligoadenylate synthetase 40 kDa splice variant(p40 subunit) (NCBI Accession Number M33863).

[0034] SEQ ID NO: 12 is the amino acid sequence for the mouse homolog ofthe 2′-5′ oligoadenylate synthetase 40 kDa splice variant (p40 subunit)(NCBI Accession Number M33863).

[0035] SEQ ID NO: 13 is the human 2′-5′ oligoadenylate synthetase 40/46kDa (p40 subunit) gene (NCBI Accession Number NM 016816).

[0036] SEQ ID NO: 14 is the human 2′-5′ oligoadenylate synthetase 69/71kDa (p69 subunit) gene (NCBI Accession Number NM_(—)002535).

[0037] SEQ ID NO: 15 is the human 2′-5′ oligoadenylate synthetase 100kDa (p100 subunit) gene (NCBI Accession Number AF063613).

[0038] SEQ ID NO: 16 is the mouse homolog of the 2′-5′ oligoadenylatesynthetase 40 kDa (p40 subunit) gene (NCBI Accession Number M33863).

[0039] SEQ ID NO: 17 is a phosphorothioate antisense oligonucleotide(ODN) designed against the p40 subunit of 2′-5′ oligoadenylatesynthetase.

[0040] SEQ ID NO: 18 is an ODN designed against the p69 subunit of 2′-5′oligoadenylate synthetase.

[0041] SEQ ID NO: 19 is a scramble of the antisense sequence to p40,i.e., identical in base composition.

[0042] SEQ ID NO: 20 is a scramble of the antisense sequence to p69.

DETAILED DISCLOSURE OF THE INVENTION

[0043] The subject invention concerns a method of inhibiting an RNAvirus infection within a patient by increasing the endogenous levels of2-5 oligoadenylate synthetase (2-5 AS) activity within the patient.Preferably, the endogenous levels of the 2-5 AS p40 subunit (e.g., 40kDa, 42 kDA, 46 kDa, or other splice variant), p69 subunit (e.g., 69kDa, 71 kDa, or other splice variant), p00 subunit, or combinationsthereof, are increased within the patient.

[0044] The present inventors have determined that overexpression of the2-5AS, or catalytically active fragments thereof, causes a reduction inepithelial cell damage, reduction in infiltration of mononuclear cellsin the peribronchiolar and perivascular regions, and reduction in thethickening of the septa in the lungs of patients suffering fromrespiratory RNA viruses, such as respiratory syncytial virus (RSV).Levels of chemokines, such as MIP1-α, are also reduced uponoverexpression of 2-5AS.

[0045] Infections from members of the family paramyxoviridae thatproduce double-stranded RNA as a requirement of replication can beprevented or treated using the present invention. Thus, infections bymembers of the genera paramyxovirus, morbillivirus, rubulavirus,pnuemovirus, and others can be inhibited in humans and animals. Examplesof RNA viruses that produce double-stranded RNA during intermediatereplication and which infect humans include, but are not limited to,respiratory syncytial virus (RSV), Rhinovirus, Vaccinia, Reovirus, HIV,EMCV, Hepatitis B, and Hepatitis C. Examples of RNA viruses that infectanimals and produce double-stranded RNA during intermediate replicationinclude, but are not limited to, bovine respiratory syncytial virus(BRSV), which infect cattle, sheep, and goats; Measles virus; Sendaivirus; Parainfluenza 1, 2, and 3; Mumps virus, Simian virus; andNewcastle virus. Infections caused by coronavirus (such as thatresponsible for severe acute respiratory syndrome (SARS)), rotavirus,parainfluenza virus, West Nile virus, as well as other viruses in whichinterferon actively inhibits viral replication can be inhibited usingthe methods and compositions of the present invention.

[0046] In one aspect, the subject invention concerns a method oftreating or preventing an RNA virus infection within a patient byincreasing the in vivo concentration of 2-5 AS or a catalytically activefragment thereof within the patient, thereby inhibiting the RNA virusinfection. Preferably, the methods of the present invention do notinvolve administration of interferon or a polynucleotide encodinginterferon, such as interferon-alpha (IFN-a), interferon-beta (IFN-β),or interferon-gamma (IFN-γ), or the administration of such IFNpolypeptides. Thus, the methods and compositions of the presentinvention are directed to increasing the in vivo concentration of 2-5 ASor a catalytically active fragment thereof, which is an IFN-γ-induceddownstream molecule. Advantageously, the methods and compositions of thepresent invention exhibit an antiviral effect without the adverseeffects associated with IFN-γ.

[0047] The in vivo concentration of the 2-5 AS, or a catalyticallyactive fragment thereof, can be increased, for example, by exogenousadministration of the 2-5 AS polypeptide, or a catalytically activefragment of the polypeptide. Preferably, the in vivo concentration ofthe 2-5 AS polypeptide or catalytically active fragment is increased byincreasing or up-regulating the functional expression of the nucleotidesequence encoding 2-5 AS or at least one catalytically active fragmentthereof, such as the p40, p69, or p100 subunits, as gene therapy. Morepreferably, a nucleotide sequence encoding 2-5 AS or at least onecatalytically active fragment thereof can be administered to a patientand expressed in order to increase the endogenous level of 2-5 ASenzymatic activity within the patient. For example, at least onenucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 14, 15, 16, and 17, or a catalytically activefragment thereof, can be administered to the patient. The nucleotidesequence can be administered to a patient's cells in vivo or in vitro(including ex vivo, genetically modifying the patient's own cells exvivo and subsequently administering the modified cells back into thepatient).

[0048] In another aspect of the invention, 2-5 AS polypeptide, or atleast one catalytically active fragment thereof, is administered to apatient in order to increase the antiviral function of 2-5 AS within thepatient. Preferably, the polypeptides utilized are those disclosedherein. The polypeptides can comprise catalytically active fragments ofthe full-length 2-5 AS amino acid sequence, such as the p40, p69, orp100 subunits, including splice variants of these subunits, or mammalianhomologs of these subunits (e.g., the p46 isoform of OAS-1; accessionnumber NP_(—)058132.1), such as murine homologs. For example, thepolypeptides can comprise one or more amino acid sequences set forthherein as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15 or 16, orcatalytically active fragments of these amino acid sequences.

[0049] Various means for delivering polypeptides to a cell can beutilized to carry out the methods of the subject invention. For example,protein transduction domains (PTDs) can be fused to the polypeptide,producing a fusion polypeptide, in which the PTDs are capable oftransducing the polypeptide cargo across the plasma membrane (Wadia, J.S. and Dowdy, S. F., Curr. Opin. Biotechnol., 2002, 13(1)52-56).Examples of PTDs include the Drosophila homeotic transcription proteinantennapedia (Antp), the herpes simples virus structural protein VP22,and the human immuno-deficiency virus 1 (HIV-1) transcriptionalactivator Tat protein.

[0050] According to the method of RNA virus inhibition of the subjectinvention, recombinant cells can be administered to a patient, whereinthe recombinant cells have been genetically modified to express the geneencoding 2-5 AS or at least one catalytically active fragment thereof,such as the p40, p69, or p100 subunits of 2-5 AS. If the cells to begenetically modified already express a gene encoding 2-5 AS, the geneticmodification can serve to enhance or increase expression of the geneencoding 2-5 AS or a catalytically active fragment of 2-5 AS beyond thenormal or constitutive amount (e.g., “overexpression”).

[0051] The method of RNA virus inhibition of the subject invention canbe used to treat a patient suffering from an RNA virus infection, or asa preventative of RNA virus infection (i.e., prophylactic treatment).According to the methods of the subject invention, various othercompounds, such as antiviral compounds, can be administered inconjunction with (before, during, or after) increasing the in vivoconcentrations of 2-5 AS or at least one catalytically active fragmentwithin the patient. Various compositions and methods for preventing ortreating RNA virus infection can be used in conjunction with thecompositions and methods of the subject invention, such as thosedescribed in U.S. Pat. No. 6,489,306, filed Feb. 23, 1999, and U.S.published patent application Serial No. 2003/00068333, filed Feb. 12,2002, which are incorporated herein by reference in their entirety. Forexample, nucleotide sequences encoding 2-5 AS or at least onecatalytically active fragment thereof can be conjugated with chitosan, abiodegradable, human-friendly cationic polymer that increases mucosalabsorption of the gene expression vaccine without any adverse effects,as described in U.S. published patent application Serial No.2003/00068333.

[0052] The nucleotide sequence can be formulated in the form ofnanospheres with chitosan. Chitosan allows increased bioavailability ofthe DNA because of protection from degradation by serum nucleases in thematrix and thus has great potential as a mucosal gene delivery system,for example. Chitosan exhibits various beneficial effects, such asanticoagulant activity, wound-healing properties, and immunostimulatoryactivity, and is capable of modulating immunity of the mucosa andbronchus-associated lymphoid tissue.

[0053] Nucleotide, polynucleotide, or nucleic acid sequences(s) areunderstood to mean, according to the present invention, either adouble-stranded DNA, a single-stranded DNA, products of transcription ofthe said DNAs (e.g., RNA molecules), or corresponding RNA molecules thatare not products of transcription. It should also be understood that thepresent invention does not relate to the genomic nucleotide sequencesencoding 2-5 AS or catalytically active fragments thereof in theirnatural/native environment or natural/native state. The nucleic acid,polynucleotide, or nucleotide sequences of the invention have beenisolated, purified (or partially purified), by separation methodsincluding, but not limited to, ion-exchange chromatography, molecularsize exclusion chromatography, affinity chromatography, or by geneticengineering methods such as amplification, cloning or subcloning.

[0054] Optionally, the polynucleotide sequence encoding 2-5 AS orcatalytically active fragment thereof can also contain one or morepolynucleotides encoding heterologous polypeptide sequences (e.g., tagsthat facilitate purification of the polypeptides of the invention (see,for example, U.S. Pat. No. 6,342,362, hereby incorporated by referencein its entirety; Altendorf et al. [1999-WWW, 2000] “Structure andFunction of the F₀ Complex of the ATP Synthase from Escherichia coli,”J. of Experimental Biology 203:19-28, The Co. of Biologists, Ltd., G.B.; Baneyx [1999] “Recombinant Protein Expression in Escherichia coli,”Biotechnology 10:411-21, Elsevier Science Ltd.; Eihauer et al. [2001]“The FLAG Peptide, a Versatile Fusion Tag for the Purification ofRecombinant Proteins,” J. Biochem Biophys Methods 49:455-65; Jones etal. [1995] J. Chromatography 707:3-22; Jones et al. [1995] “CurrentTrends in Molecular Recognition and Bioseparation,” J. of ChromatographyA. 707:3-22, Elsevier Science B. V.; Margolin [2000] “Green FluorescentProtein as a Reporter for Macromolecular Localization in BacterialCells,” Methods 20:62-72, Academic Press; Puig et al. [2001] “The TandemAffinity Purification (TAP) Method: A General Procedure of ProteinComplex Purification,” Methods 24:218-29, Academic Press; Sassenfeld[1990] “Engineering Proteins for Purification,” TibTech 8:88-93;Sheibani [1999] “Prokaryotic Gene Fusion Expression Systems and TheirUse in Structural and Functional Studies of Proteins,” Prep. Biochem. &Biotechnol. 29(1):77-90, Marcel Dekker, Inc.; Skerra et al [1999]“Applications of a Peptide Ligand for Streptavidin: the Strep-tag”,Biomolecular Engineering 16:79-86, Elsevier Science, B. V.; Smith [1998]“Cookbook for Eukaryotic Protein Expression: Yeast, Insect, and PlantExpression Systems,” The Scientist 12(22):20; Smyth et al. [2000]“Eukaryotic Expression and Purification of Recombinant ExtracellularMatrix Proteins Carrying the Strep II Tag”, Methods in MolecularBiology, 139:49-57; Unger [1997] “Show Me the Money: ProkaryoticExpression Vectors and Purification Systems,” The Scientist 11(17):20,each of which is hereby incorporated by reference in their entireties),or commercially available tags from vendors such as such as STRATAGENE(La Jolla, Calif.), NOVAGEN (Madison, Wis.), QIAGEN, Inc., (Valencia,Calif.), or INVITROGEN (San Diego, Calif.).

[0055] Other aspects of the invention provide vectors containing one ormore of the polynucleotides of the invention, such as vectors containingnucleotides encoding 2-5 AS or catalytically active fragments of 2-5 AS,such as the p40 and/or p69 subunits. The vectors can be vaccine,replication, or amplification vectors. In some embodiments of thisaspect of the invention, the polynucleotides are operably associatedwith regulatory elements capable of causing the expression of thepolynucleotide sequences. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, lentiviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations of the aforementioned vector sources, such as those derivedfrom plasmid and bacteriophage genetic elements (e.g., cosmids andphagemids). Preferably, the vector is an adenoaviral vector oradeno-associated virus vector.

[0056] As indicated above, vectors of this invention can also compriseelements necessary to provide for the expression and/or the secretion of2-5 AS, or a catalytically active fragment thereof, encoded by thenucleotide sequences of the invention in a given host cell. The vectorcan contain one or more elements selected from the group consisting of apromoter sequence, signals for initiation of translation, signals fortermination of translation, and appropriate regions for regulation oftranscription. In certain embodiments, the vectors can be stablymaintained in the host cell and can, optionally, contain signalsequences directing the secretion of translated protein. Otherembodiments provide vectors that are not stable in transformed hostcells. Vectors can integrate into the host genome or beautonomously-replicating vectors.

[0057] In a specific embodiment, a vector comprises a promoter operablylinked to a 2-5 AS-encoding nucleic acid sequence (or a catalyticallyactive fragment thereof), one or more origins of replication, and,optionally, one or more selectable markers (e.g., an antibioticresistance gene). Non-limiting exemplary vectors for the expression ofthe polypeptides of the invention include pBr-type vectors, pET-typeplasmid vectors (PROMEGA), pBAD plasmid vectors (INVITROGEN), and pVAXplasmid vectors (INVITROGEN), or others provided in the examples below.Furthermore, vectors according to the invention are useful fortransforming host cells for the cloning or expression of the nucleotidesequences of the invention.

[0058] Promoters which may be used to control expression include, butare not limited to, the CMV promoter, the SV40 early promoter region(Bemoist and Chambon [1981] Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al.[1980] Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal. [1981] Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al. [1982] Nature296:39-42); prokaryotic vectors containing promoters such as theβ-lactamase promoter (Villa-Kamaroff, et al. [1978] Proc. Natl. Acad.Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al. [1983] Proc.Natl. Acad. Sci. USA 80:21-25); the lung specific promoters such assurfactant protein B promoter (Venkatesh et al., Am. J. Physiol. 268(Lung Cell Mol. Physiol. 12):L674-L682, 1995); see also, “UsefulProteins from Recombinant Bacteria” in Scientific American, 1980,242:74-94; plant expression vectors comprising the nopaline synthetasepromoter region (Herrera-Estrella et al. [1983] Nature 303:209-213) orthe cauliflower mosaic virus 35S RNA promoter (Gardner, et al. [1981]Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzymeribulose biphosphate carboxylase (Herrera-Estrella et al. [1984] Nature310:115-120); promoter elements from yeast or fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, and/or the alkaline phosphatase promoter.

[0059] The subject invention also provides for “homologous” or“modified” nucleotide sequences. Modified nucleic acid sequences will beunderstood to mean any nucleotide sequence obtained by mutagenesisaccording to techniques well known to persons skilled in the art, andexhibiting modifications in relation to the normal sequences. Forexample, mutations in the regulatory and/or promoter sequences for theexpression of a polypeptide that result in a modification of the levelof expression of a polypeptide according to the invention provide for a“modified nucleotide sequence”. Likewise, substitutions, deletions, oradditions of nucleic acid to the polynucleotides of the inventionprovide for “homologous” or “modified” nucleotide sequences. In variousembodiments, “homologous” or “modified” nucleic acid sequences havesubstantially the same biological activity as the native (naturallyoccurring) 2-5 AS or subunit thereof. A “homologous” or “modified”nucleotide sequence will also be understood to mean a subunit or asplice variant of the polynucleotides of the instant invention or anynucleotide sequence encoding a “modified polypeptide” as defined below.

[0060] A homologous nucleotide sequence, for the purposes of the presentinvention, encompasses a nucleotide sequence having a percentageidentity with the bases of the nucleotide sequences of between at least(or at least about) 20.00% to 99.99% (inclusive), and which encodes acatalytically active polypeptide. The aforementioned range of percentidentity is to be taken as including, and providing written descriptionand support for, any fractional percentage, in intervals of 0.01%,between 20.00% and 99.99%. These percentages are purely statistical anddifferences between two nucleic acid sequences can be distributedrandomly and over the entire sequence length.

[0061] In various embodiments, homologous sequences exhibiting apercentage identity with the bases of the nucleotide sequences of thepresent invention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percentidentity with the polynucleotide sequences of the instant invention.

[0062] Both protein and nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW(Pearson and Lipman [1988] Proc. Natl. Acad. Sci. USA 85(8):2444-2448;Altschul et al. [1990] J. Mol. Biol. 215(3):403-410; Thompson et al.[1994] Nucleic Acids Res. 22(2):4673-4680; Higgins et al. [1996] MethodsEnzymol. 266:383-402; Altschul et al. [1990] J. Mol. Biol.215(3):403-410; Altschul et al. [1993] Nature Genetics 3:266-272).

[0063] Nucleotide sequences encoding polypeptides with enhanced 2-5 AScatalytic activity can be obtained by “gene shuffling” (also referred toas “directed evolution”, and “directed mutagenesis”), and used in thecompositions and methods of the present invention. Gene shuffling is aprocess of randomly recombining different sequences of functional genes(recombining favorable mutations in a random fashion) (U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; and 5,837,458). Thus, proteinengineering can be accomplished by gene shuffling, random complexpermutation sampling, or by rational design based on three-dimensionalstructure and classical protein chemistry (Cramer et al., Nature,391:288-291, 1998; and Wulff et al., The Plant Cell, 13:255-272, 2001).

[0064] The subject invention also provides nucleotide sequencescomplementary to any of the polynucleotide sequences disclosed herein.Thus, the invention is understood to include any DNA whose nucleotidesare complementary to those of 2-5 AS polynucleotide sequence of theinvention, or catalytically active fragments thereof, and whoseorientation is reversed (e.g., an antisense sequence).

[0065] The present invention further provides catalytically activefragments of the 2-5 AS polynucleotide sequences, includingcatalytically active fragments of the 2-5 AS subunit nucleotidesequences, provided herein. Representative fragments of thepolynucleotide sequences according to the invention will be understoodto mean any nucleotide fragment having at least 8 or 9 successivenucleotides, preferably at least 12 successive nucleotides, and stillmore preferably at least 15 or at least 20 successive nucleotides of thesequence from which it is derived. The upper limit for such fragments isthe total number of polynucleotides found in the full-length sequence(or, in certain embodiments, of the full length open reading frame (ORF)identified herein). It is understood that, optionally, such fragmentsrefer only to portions of the disclosed polynucleotide sequences thatare not listed in a publicly available database or prior art references.However, it should be understood that with respect to the method forinhibiting RSV of the subject invention, disclosed nucleotides (andpolypeptides encoded by such nucleotides) that are listed in a publiclyavailable database or prior art reference can also be utilized. Forexample, nucleotide sequences that are 2-5 AS p40 or p69 subunithomologs, or fragments thereof, which have been previously identified,can be utilized to carry out the method for inhibiting RNA virusinfection of the subject invention.

[0066] In other embodiments, fragments contain from one nucleotide lessthan the full length 2-5 AS enzyme, or from one nucleotide less than acatalytically active subunit thereof, such as p40 or p69 subunitpolynucleotide CDS sequences (e.g., 1,203 and 1,207 nucleotides for the40 kDa splice variant and 46 kDa splice variant, respectively; and 2063and 2,168 nucleotides for the 69 kDa splice variant and 71 kDa splicevariant, respectively) to fragments containing the smallest number ofnucleotides encoding a polypeptide that retains at least some 2-5 ASenzymatic activity.

[0067] Among these representative fragments, those capable ofhybridizing under stringent conditions with a nucleotide sequenceencoding 2-5 AS or subunits thereof are preferred. Conditions of high orintermediate stringency are provided infra and are chosen to allow forhybridization between two complementary DNA fragments. Hybridizationconditions for a polynucleotide of about 1,000 to 3,000 bases in sizewill be adapted by persons skilled in the art for larger- orsmaller-sized oligonucleotides, according to methods well known in theart (see, for example, Sambrook et al. [1989] Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp.9.47-9.57).

[0068] The subject invention also provides detection probes (e.g.,fragments of the disclosed polynucleotide sequences) for hybridizationwith a target sequence or an amplicon generated from the targetsequence. Such a detection probe will advantageously have as sequence asequence of at least 9, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 nucleotides. Alternatively, detectionprobes can comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127 and up to, for example, 1,203 consecutivenucleotides, 1,207 consecutive nucleotides, 2,064 consecutivenucleotides, 2,186 consecutive nucleotides, 3,264 consecutivenucleotides, and 1,104 consecutive nucleotides of those disclosedherein, which correspond, respectively, to the human 40 kDa splicevariant of the 2-5AS p40 subunit (SEQ ID NO:1), human 46 kDa splicevariant 2-5AS p40 subunit (SEQ ID NO:3), human 69 kDa splice variant ofthe 2-5AS p69 subunit (SEQ ID NO:5), human 71 kDa splice variant of the2-5AS p69 subunit (SEQ ID NO:7), human p100 subunit (SEQ ID NO:9), andthe mouse homolog of the 2-5AS p40 subunit (SEQ ID NO: 11). Thedetection probes can also be used as labeled probe or primer in thesubject invention. Labeled probes or primers are labeled with aradioactive compound or with another type of label. Alternatively,non-labeled nucleotide sequences may be used directly as probes orprimers; however, the sequences are generally labeled with a radioactiveelement (³²P, 35S, ³H, ¹²⁵I) or with a molecule such as biotin,acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine, or fluoresceinto provide probes that can be used in numerous applications.

[0069] The nucleotide sequences according to the invention may also beused in analytical systems, such as DNA chips. DNA chips and their usesare well known in the art and (see for example, U.S. Pat. Nos.5,561,071; 5,753,439; 6,214,545; Schena el al. [1996] BioEssays18:427-431; Bianchi et al. [1997] Clin. Diagn. Virol. 8:199-208; each ofwhich is hereby incorporated by reference in their entireties) and/orare provided by commercial vendors such as AFFYMETRIX, Inc. (SantaClara, Calif.).

[0070] Various degrees of stringency of hybridization can be employed.The more severe the conditions, the greater the complementarity that isrequired for duplex formation. Severity of conditions can be controlledby temperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller, G. H., M. M. Manak [1987] DNA Probes,Stockton Press, New York, N.Y., pp. 169-170.

[0071] By way of example, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes can be performed by standardmethods (Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York). In general, hybridization andsubsequent washes can be carried out under moderate to high stringencyconditions that allow for detection of target sequences with homology tothe exemplified polynucleotide sequence. For double-stranded DNA geneprobes, hybridization can be carried out overnight at 20-25° C. belowthe melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5× Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz et al. [1983] Methods ofEnzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, NewYork 100:266-285).

[0072] T_(m)=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61(%formamide)−600/length of duplex in base pairs.

[0073] Washes are typically carried out as follows:

[0074] (1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS(low stringency wash);

[0075] (2) once at T_(m)−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS(moderate stringency wash).

[0076] For oligonucleotide probes, hybridization can be carried outovernight at 10-20° C. below the melting temperature (T_(m)) of thehybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denaturedDNA. Tm for oligonucleotide probes can be determined by the followingformula:

[0077] T_(m), (° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs et al. [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D.D. Brown [ed.], Academic Press, New York, 23:683-693).

[0078] Washes can be carried out as follows:

[0079] (1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS(low stringency wash;

[0080] 2) once at the hybridization temperature for 15 minutes in1×SSPE, 0.1% SDS (moderate stringency wash).

[0081] In general, salt and/or temperature can be altered to changestringency. With a labeled DNA fragment >70 or so bases in length, thefollowing conditions can be used:

[0082] Low: 1 or 2×SSPE, room temperature

[0083] Low: 1 or 2×SSPE, 42° C.

[0084] Moderate: 0.2× or 1×SSPE, 65° C.

[0085] High: 0.1×SSPE, 65° C.

[0086] By way of another non-limiting example, procedures usingconditions of high stringency can also be performed as follows:Pre-hybridization of filters containing DNA is carried out for 8 h toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C.,the preferred hybridization temperature, in pre-hybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Alternatively, the hybridization step can beperformed at 65° C. in the presence of SSC buffer, 1×SSC correspondingto 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can bedone at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01%Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45min. Alternatively, filter washes can be performed in a solutioncontaining 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps,the hybridized probes are detectable by autoradiography. Otherconditions of high stringency which may be used are well known in theart (see, for example, Sambrook et al. [1989] Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp.9.47-9.57; and Ausubel et al. [1989] Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y., eachincorporated herein in its entirety).

[0087] A further non-limiting example of procedures using conditions ofintermediate stringency are as follows: Filters containing DNA arepre-hybridized, and then hybridized at a temperature of 60° C. in thepresence of a 5×SSC buffer and labeled probe. Subsequently, filterswashes are performed in a solution containing 2×SSC at 50° C. and thehybridized probes are detectable by autoradiography. Other conditions ofintermediate stringency which may be used are well known in the art(see, for example, Sambrook et al. [1989] Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp.9.47-9.57; and Ausubel et al [1989] Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y., eachof which is incorporated herein in its entirety).

[0088] Duplex formation and stability depend on substantialcomplementarity between the two strands of a hybrid and, as noted above,a certain degree of mismatch can be tolerated. Therefore, the probesequences of the subject invention include mutations (both single andmultiple), deletions, insertions of the described sequences, andcombinations thereof, wherein said mutations, insertions and deletionspermit formation of stable hybrids with the target polynucleotide ofinterest. Mutations, insertions and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

[0089] It is also well known in the art that restriction enzymes can beused to obtain functional fragments of the subject DNA sequences. Forexample, Bal31 exonuclease can be conveniently used for time-controlledlimited digestion of DNA (commonly referred to as “erase-a-base”procedures). See, for example, Maniatis et al. [1982] Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Wei et al.[1983] J Biol. Chem. 258:13006-13512. The nucleic acid sequences of thesubject invention can also be used as molecular weight markers innucleic acid analysis procedures.

[0090] The invention also provides host cells transformed by apolynucleotide according to the invention and the production of 2-5 ASor a catalytically active fragment thereof, by the transformed hostcells. In some embodiments, transformed cells comprise an expressionvector containing 2-5 AS nucleotide sequences or a catalytically activefragment thereof. Other embodiments provide for host cells transformedwith nucleic acids. Yet other embodiments provide transformed cellscomprising an expression vector containing fragments of 2-5 AS p40and/or p69 subunit nucleotide sequences. Transformed host cellsaccording to the invention are cultured under conditions allowing thereplication and/or the expression of the 2-5 AS nucleotide sequence or acatalytically active fragment thereof, such as the p40 and/or p69subunits. Expressed polypeptides are recovered from culture media andpurified, for further use, according to methods known in the art.

[0091] The host cell may be chosen from eukaryotic or prokaryoticsystems, for example bacterial cells (Gram negative or Gram positive),yeast cells, animal cells, human cells, plant cells, and/or insect cellsusing baculovirus vectors. In some embodiments, the host cell forexpression of the polypeptides include, and are not limited to, thosetaught in U.S. Pat. Nos. 6,319,691; 6,277,375; 5,643,570; 5,565,335;Unger [1997] The Scientist 11(17):20; or Smith [1998] The Scientist12(22):20, each of which is incorporated by reference in its entirety,including all references cited within each respective patent orreference. Other exemplary, and non-limiting, host cells includeStaphylococcus spp., Enterococcus spp., E. coli, and Bacillus subtilis;fungal cells, such as Streptomyces spp., Aspergillus spp., S.cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hanselapolymorpha, Kluveromyces lactis, and Yarrowia lipolytica; insect cellssuch as Drosophila S2 and Spodoptera Sf9 cells; animal cells such asCHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plantcells. A great variety of expression systems can be used to produce the2-5 AS polypeptides or catalytically active fragments thereof andencoding polynucleotides can be modified according to methods known inthe art to provide optimal codon usage for expression in a particularexpression system.

[0092] Furthermore, a host cell strain may be chosen that modulates theexpression of the inserted sequences, modifies the gene product, and/orprocesses the gene product in the specific fashion. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered polypeptide may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation) ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system can be used toproduce an unglycosylated core protein product whereas expression inyeast will produce a glycosylated product. Expression in mammalian cellscan be used to provide “native” glycosylation of a heterologous protein.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

[0093] Nucleic acids and/or vectors encoding 2-5 AS, or catalyticallyactive fragments thereof, such as the p40 and/or p69 subunits, can beintroduced into host cells by well-known methods, such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transfection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection (see, for example, Sambrook et al. [1989] MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

[0094] The subject invention also provides for the expression of the 2-5AS p40 or p69 subunit, derivative, or a analogue (e.g., a splicevariant) encoded by a polynucleotide sequence disclosed herein.Alternatively, the invention provides for the expression of apolynucleotide encoding a fragment of a 2-5 AS p40 or p69 subunit. Ineither embodiment, the disclosed sequences can be regulated by a secondnucleic acid sequence so that the polypeptide or fragment is expressedin a host transformed with a recombinant DNA molecule according to thesubject invention. For example, expression of a protein or peptide maybe controlled by any promoter/enhancer element known in the art.

[0095] In the context of the instant invention, the terms polypeptide,peptide and protein are used interchangeably. Likewise, the termsanalogue and homologous are also used interchangeably. It should beunderstood that the invention does not relate to the polypeptides innatural form or native environment. Peptides and polypeptides accordingto the invention have been isolated or obtained by purification fromnatural sources (or their native environment), chemically synthesized,or obtained from host cells prepared by genetic manipulation (e.g., thepolypeptides, or fragments thereof, are recombinantly produced by hostcells). Polypeptides according to the instant invention may also containnon-natural amino acids, as will be described below.

[0096] “Analogues” or “homologous” polypeptides will be understood todesignate the polypeptides containing, in relation to the nativepolypeptide, modifications such as deletion, addition, or substitutionof at least one amino acid, truncation, extension, or the addition ofchimeric heterologous polypeptides. Optionally, “analogues” or“homologous” polypeptides can contain a mutation or post-translationalmodifications. Among the “analogues” or “homologous” polypeptides, thosewhose amino acid sequence exhibits 20.00% to 99.99% (inclusive) identityto the native polypeptide sequence are preferred. The aforementionedrange of percent identity is to be taken as including, and providingwritten description and support for, any fractional percentage, inintervals of 0.01%, between 50.00% and, up to, including 99.99%. Thesepercentages are purely statistical and differences between twopolypeptide sequences can be distributed randomly and over the entiresequence length.

[0097] “Analogues” or “homologous” polypeptide sequences exhibiting apercentage identity with the human 2-5 AS polypeptides, or subunitsthereof, can alternatively have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 91, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent identity with the polypeptide sequences of the instantinvention. The expression equivalent amino acid is intended here todesignate any amino acid capable of being substituted for one of theamino acids in the basic structure without, however, essentiallymodifying the biological activities of the corresponding peptides and asprovided below.

[0098] By way of example, amino acid substitutions can be carried outwithout resulting in a substantial modification of the biologicalactivity of the corresponding modified polypeptides; for example, thereplacement of leucine with valine or isoleucine; aspartic acid withglutamic acid; glutamine with asparagine; arginine with lysine; and thereverse substitutions can be performed without substantial modificationof the biological activity of the polypeptides.

[0099] The subject invention also provides catalytically activefragments of the 2-5 AS polypeptide, and catalytically active fragmentsof the 2-5 AS subunits, according to the invention, which are capable ofeliciting an immune response against RSV. The immune response canprovide components (either antibodies or components of the cellularimmune response (e.g., B-cells, helper, cytotoxic, and/or suppressorT-cells) reactive with the catalytically active fragment of thepolypeptide, the intact, full length, unmodified polypeptide, or boththe catalytically active fragment of the polypeptide and the intact,full length, unmodified polypeptides.

[0100] Catalytically active fragments according to the invention cancomprise from five (5) amino acids to one amino acid less than the fulllength of any polypeptide sequence provided herein. For example,fragments comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, and up to one amino acid less than the fulllength amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, and SEQ ID NO:12, are provided herein.

[0101] Fragments, as described herein, can be obtained by cleaving thepolypeptides of the invention with a proteolytic enzyme (such astrypsin, chymotrypsin, or collagenase) or with a chemical reagent, suchas cyanogen bromide (CNBr). Alternatively, polypeptide fragments can begenerated in a highly acidic environment, for example at pH 2.5. Suchpolypeptide fragments may be equally well prepared by chemical synthesisor using hosts transformed with an expression vector containing nucleicacids encoding polypeptide fragments according to the invention. Thetransformed host cells contain a nucleic acid and are cultured accordingto well-known methods; thus, the invention allows for the expression ofthese fragments, under the control of appropriate elements forregulation and/or expression of the polypeptide fragments.

[0102] Modified polypeptides according to the invention are understoodto designate a polypeptide obtained by variation in the splicing oftranscriptional products of the 2-5 AS gene, genetic recombination, orby chemical synthesis as described below. Modified polypeptides containat least one modification in relation to the normal polypeptidesequence. These modifications can include the addition, substitution,deletion of amino acids contained within the polypeptides of theinvention.

[0103] Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution does not materiallyalter the biological activity of the polypeptide. For example, the classof nonpolar amino acids include Ala, Val, Leu, Ile, Pro, Met, Phe, andTrp; the class of uncharged polar amino acids includes Gly, Ser, Thr,Cys, Tyr, Asn, and Gln; the class of acidic amino acids includes Asp andGlu; and the class of basic amino acids includes Lys, Arg, and His. Insome instances, non-conservative substitutions can be made where thesesubstitutions do not significantly detract from the biological activityof the polypeptide.

[0104] In order to extend the life of the polypeptides of the invention,it may be advantageous to use non-natural amino acids, for example inthe D form, or alternatively amino acid analogs, such assulfur-containing forms of amino acids. Alternative means for increasingthe life of polypeptides can also be used in the practice of the instantinvention. For example, polypeptides of the invention, and fragmentsthereof, can be recombinantly modified to include elements that increasethe plasma, or serum half-life of the polypeptides of the invention.These elements include, and are not limited to, antibody constantregions (see for example, U.S. Pat. No. 5,565,335, hereby incorporatedby reference in its entirety, including all references cited therein),or other elements such as those disclosed in U.S. Pat. Nos. 6,319,691;6,277,375; or 5,643,570, each of which is incorporated by reference inits entirety, including all references cited within each respectivepatent. Alternatively, the 2-5 AS polynucleotides, or catalyticallyactive fragments thereof, used in the instant invention can berecombinantly fused to elements that are useful in the preparation ofimmunogenic constructs for the purposes of vaccine formulation orelements useful for the isolation of the polypeptides of the invention.

[0105] The polypeptides, fragments, and immunogenic fragments of theinvention may further contain linkers that facilitate the attachment ofthe fragments to a carrier molecule for delivery or diagnostic purposes.The linkers can also be used to attach fragments according to theinvention to solid support matrices for use in affinity purificationprotocols. In this aspect of the invention, the linkers specificallyexclude, and are not to be considered anticipated, where the fragment isa subsequence of another peptide, polypeptide, or protein as identifiedin a search of protein sequence databases as indicated in the precedingparagraph. In other words, the non-identical portions of the otherpeptide, polypeptide, or protein is not considered to be a “linker” inthis aspect of the invention. Non-limiting examples of “linkers”suitable for the practice of the invention include chemical linkers(such as those sold by Pierce, Rockford, Ill.), peptides that allow forthe connection of the immunogenic fragment to a carrier molecule (see,for example, linkers disclosed in U.S. Pat. Nos. 6,121,424; 5,843,464;5,750,352; and 5,990,275, hereby incorporated by reference in theirentirety). In various embodiments, the linkers can be up to 50 aminoacids in length, up to 40 amino acids in length, up to 30 amino acids inlength, up to 20 amino acids in length, up to 10 amino acids in length,or up to 5 amino acids in length.

[0106] In other specific embodiments, the 2-5 AS polypeptide or 2-5 ASsubunit polypeptide, peptides, derivatives, or analogs thereof may beexpressed as a fusion, or chimeric protein product (comprising theprotein, fragment, analog, or derivative joined via a peptide bond to aheterologous protein sequence (e.g., a different protein)). Such achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other bymethods known in the art, in the proper coding frame, and expressing thechimeric product by methods commonly known in the art (see, for example,U.S. Pat. No. 6,342,362, hereby incorporated by reference in itsentirety; Altendorf et al. [999-WWW, 2000] “Structure and Function ofthe F₀ Complex of the ATP Synthase from Escherichia coli,” J. ofExperimental Biology 203:19-28, The Co. of Biologists, Ltd., G. B.;Baneyx [1999] “Recombinant Protein Expression in Escherichia coli,”Biotechnology 10:411-21, Elsevier Science Ltd.; Eihauer et al. [2001]“The FLAG Peptide, a Versatile Fusion Tag for the Purification ofRecombinant Proteins,” J. Biochem Biophys Methods 49:455-65; Jones etal. [1995] J. Chromatography 707:3-22; Jones et al. [1995] “CurrentTrends in Molecular Recognition and Bioseparation,” J. Chromatography A.707:3-22, Elsevier Science B. V.; Margolin [2000] “Green FluorescentProtein as a Reporter for Macromolecular Localization in BacterialCells,” Methods 20:62-72, Academic Press; Puig et al. [2001] “The TandemAffinity Purification (TAP) Method: A General Procedure of ProteinComplex Purification,” Methods 24:218-29, Academic Press; Sassenfeld[1990] “Engineering Proteins for Purification,” TibTech 8:88-93;Sheibani [1999] “Prokaryotic Gene Fusion Expression Systems and TheirUse in Structural and Functional Studies of Proteins,” Prep. Biochem. &Biotechnol. 29(1):77-90, Marcel Dekker, Inc.; Skerra et al. [1999]“Applications of a Peptide Ligand for Streptavidin: The Strep-tag”,Biomolecular Engineering 16:79-86, Elsevier Science, B. V.; Smith [1998]“Cookbook for Eukaryotic Protein Expression: Yeast, Insect, and PlantExpression Systems,” The Scientist 12(22):20; Smyth et al. [2000]“Eukaryotic Expression and Purification of Recombinant ExtracellularMatrix Proteins Carrying the Strep II Tag”, Methods in MolecularBiology, 139:49-57; Unger [1997] “Show Me the Money: ProkaryoticExpression Vectors and Purification Systems,” The Scientist 11(17):20,each of which is hereby incorporated by reference in their entireties).Alternatively, such a chimeric product may be made by protein synthetictechniques, e.g., by use of a peptide synthesizer. Fusion peptides cancomprise polypeptides of the subject invention and one or more proteintransduction domains, as described above. Such fusion peptides areparticularly useful for delivering the cargo polypeptide through thecell membrane.

[0107] Increasing the amount of 2-5 AS enzymatic activity (e.g., p40,p69, and/or p100 subunit activity) within a tissue is useful inpreventing an RNA virus infection, or in treating an existing RNA virusinfection. Thus, according to the methods of the subject invention, theamount of 2-5 AS activity can be increased within a tissue by directlyadministering the 2-5 AS polypeptide or a catalytically active fragmentthereof to a patient suffering from or susceptible to an RNA virusinfection (such as exogenous delivery of the 2-5 AS p40, p69, and/orp100 subunit polypeptide) or by indirect or genetic means (such asdelivery of a nucleotide sequence encoding the 2-5 AS polypeptide or acatalytically active fragment thereof, or upregulating the endogenous2-5 AS polypeptide activity).

[0108] As used herein, the term “administration” or “administering”refers to the process of delivering an agent to a patient, wherein theagent directly or indirectly increases 2-5 AS enzymatic function withinthe patient and, preferably, at the target site. The process ofadministration can be varied, depending on the agent, or agents, and thedesired effect. Thus, wherein the agent is genetic material, such asDNA, the process of administration involves administering a DNA encoding2-5 AS, or a catalytically active fragment thereof, to a patient in needof such treatment. Administration can be accomplished by any meansappropriate for the therapeutic agent, for example, by parenteral,mucosal, pulmonary, topical, catheter-based, or oral means of delivery.Parenteral delivery can include for example, subcutaneous intravenous,intrauscular, intra-arterial, and injection into the tissue of an organ,particularly tumor tissue. Mucosal delivery can include, for example,intranasal delivery. According to the method of the present invention, anucleotide sequence encoding the 2-5 AS or catalytically active fragmentis preferably administered into the airways of a patient, i.e., nose,sinus, throat, lung, for example, as nose drops, by nebulization,vaporization, or other methods known in the art. Oral or intranasaldelivery can include the administration of a propellant. Pulmonarydelivery can include inhalation of the agent. Catheter-based deliverycan include delivery by iontropheretic catheter-based delivery. Oraldelivery can include delivery of a coated pill, or administration of aliquid by mouth. Administration can generally also include delivery witha pharmaceutically acceptable carrier, such as, for example, a buffer, apolypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or alipid. Gene therapy protocol is also considered an administration inwhich the therapeutic agent is a polynucleotide capable of accomplishinga therapeutic goal when expressed as a transcript or a polypeptide intothe patient. Further information concerning applicable gene therapyprotocols is provided in the examples disclosed herein.

[0109] The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Science (Martin EW [1995] EastonPennsylavania, Mack Publishing Company, 19^(th) ed.) describesformulations which can be used in connection with the subject invention.Formulations suitable for parenteral administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

[0110] Therapeutically effective and optimal dosage ranges for 2-5 AS orcatalytically active fragments thereof can be determined using methodsknown in the art. Guidance as to appropriate dosages to achieve ananti-viral effect is provided from the exemplified assays disclosedherein.

[0111] As used herein, the term “catalytic activity” with respect tofragments, analogues, and hornologs of the 2-5 AS polypeptide, or tofragments, analogues, and homologues of nucleotide sequences encodingthe 2-5 AS polypeptide, refers to 2′-5′ oligoadenylate synthetaseactivity. As used herein, “2′-5′ oligoadenylate synthetase activity”refers to polymerization of ATP to produce 2′-5′ linked oligoadenylates,which in turn, activate a latent ribonuclease, RNase L, that degradesRNAs (see, for example, Katze et al., Nat. Rev. Immunol., September2002, 2(9):675-687; Justesen et al., Cell Mol. Life Sci., 57:1593-1612,2000; Hartmann et al., J. Bio. Chem., 273(6):3236-3246, 1998; U.S. Pat.No. 5,766,864). Preferably, the catalytic activity is an amounteffective to inhibit RNA virus infection (pre-infection orpost-infection). 2′-5′ oligoadenylate synthetase activity can bedetermined directly or indirectly in vivo, or in vitro, using methodsknown in the art. Thus, cell-based assays can be utilized to determinewhether an agent, such as a nucleotide sequence or polypeptide, exhibitsthe relevant catalytic activity, and can be utilized to carry out themethod of RNA virus inhibition of the subject invention.

[0112] RNA virus infections that can be inhibited using the presentinvention include those that must produce double-stranded RNA as anintermediate step in viral replication and those viruses for whichinterferon can actively inhibit viral replication. These RNA viruses canincluded single-stranded or double-stranded RNA viruses, and havegenomes of positive (+) or negative (−) strand polarity.

[0113] The present invention further provides methods of making the hostcells, pharmaceutical compositions, and vectors described herein bycombining the various components using methods known in the art.

[0114] The term “patient”, as used herein, refers to any vertebratespecies. Preferably, the patient is of a mammalian species. Mammalianspecies which benefit from the disclosed methods of treatment include,and are not limited to, apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Human ornon-human animal patients can range in age from neonates to elderly. Thenucleotide sequences and polypeptides can be administered to patients ofthe same species or from different species. For example, mammalian,homologs can be administered to human patients.

[0115] The terms “2-5 AS p40 subunit” and “2-5 AS p40 subunitpolypeptide” are used herein interchangeably to refer to the 2′-5′oligoadenlate synthetase p40 subunit gene or its coding sequence (CDS),its polypeptide product, or a catalytically active fragment or analogueof the polypeptide product, and includes 2-5 AS p40 subunit peptidehomologs (such as mammalian orthologs (e.g., SEQ ID NOs: 11 and 12);NCBI Accession Number M33863) and isoforms, unless otherwise noted.Thus, the term includes all splice variants of the p40 subunit, such asthe 40 kDa (SEQ ID NOs: 1 and 2), 42 kDa, and 46 kDa (SEQ ID NOs:3 and4) splice variants of the 2-5 AS p40 subunit (NCBI Accession NumberNM_(—)016816).

[0116] The terms “2-5 AS p69 subunit” and “2-5 AS p69 subunitpolypeptide” are used herein interchangeably to refer to the 2′-5.′oligoadenlate synthetase. p69 subunit gene or its coding sequence (CDS),its polypeptide product, or a catalytically active fragment or analogueof the polypeptide product, and includes 2-5 AS p69 subunit peptidehomologs (such as mammalian orthologs) and isoforms, unless otherwisenoted. Thus, the term includes all splice variants of the p69 subunit,such as the 69 kDa (SEQ ID NOs:5 and 6) and 71 kDa (SEQ ID NOs:7 and 8)splice variants of the 2-5 AS p69 subunit (NCBI Accession NumberNM_(—)002535).

[0117] The terms “2-5 AS p100 subunit” and “2-5 AS p100 subunitpolypeptide” are used herein interchangeably to refer to the 2′-5′oligoadenlate synthetase p100 subunit gene or its coding sequence (CDS)(SEQ ID NO:9), its polypeptide product (SEQ ID NO:10), or acatalytically active fragment or analogue of the polypeptide product,and includes 2-5 AS p100 subunit peptide homologs (such as mammalianorthologs) and isoforms, unless otherwise noted. Thus, the term includesall splice variants of the p100 subunit (NCBI Accession NumberAF063613).

[0118] The terms “comprising”, “consisting of”, and “consistingessentially of” are defined according to their standard meaning and maybe substituted for one another throughout the instant application inorder to attach the specific meaning associated with each term.

Materials and Methods

[0119] Epithelial Cell Culture, Virus Infection and Plaque Assay. TheHEp-2 (ATCC CCL-23) cell line was obtained from the American TypeCulture Collection (Manassass, Va.) and was maintained in MinimumEssential medium with Hank's salts (MEM) supplemented with 5% fetalbovine serum (FBS) (ATLANTA BIOLOGICALS, Norcross, Ga.) at 37° C. with5% C02. Respiratory syncytial virus (RSV) A2 strain was obtained fromATCC (VR-1302) and was propagated in HEp-2 cells grown in MEM with 2%FBS on a monolayer culture. Viral stocks were prepared from infectedHEp-2 cells 5 days post infection (p.i.), stored at −700C in aliquotsand used as the viral inoculum. RSV titers were quantified by plaqueassay as described earlier (21).

[0120] MTT Cytotoxicity Assay. The effect of IFN-γ on the viability ofcells was determined using a MTT[3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide] (SIGMA,St. Louis, Mo.) cytotoxicity assay. Triplicate sets of cell monolayerswere used for each IFN-γ dose tested and for each time point. In thissystem, the mitochondrial dehydrogenase enzymes of living cells cleavethe tetrazolium ring of the yellow MTT to form purple formazan crystals,which are insoluble in aqueous solutions. The crystals were dissolved inacidified isopropanol, and the absorbance of the resulting purplesolution was spectrophotometrically measured at 540 nm. An increase ordecrease in the viable cell number results in a concomitant change inthe amount of formazan formed, indicating the degree of cytotoxicitycaused by the indicated dose of IFN-γ.

[0121] Immunoblot Analysis. IFN-γ treated cells were washed in cold PBS,pH 7.4 and scraped into PBS at various time points. The cells werecollected by centrifugation at 6000 rpm for 3 min at 4° C. and the cellpellet was suspended in a 2-pack volume of cell lysis buffer (50 mMTris-HCl, pH 7.4; 1% NP-40; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 mg/mlaprotinin, leupeptin, pepstatin) and vortexed thoroughly. The celllysate was spun at 13,000 rpm for 15′ at 4° C. to remove cellulardebris. The supernatant was collected and the protein content estimatedusing the BCA (bicinchoninic acid) assay (PIERCE, Rockford, Ill.). 30 mgof total protein was mixed with an equal volume of 2×SDS sample buffer(22) and loaded onto a 10% SDS-PAGE and run at a 30 mA constant currentfor 2 to 2.5 hours. For the detection of iNOS, the lysate of the IFN-γand LPS-stimulated murine macrophage (RAW 264.7) was loaded onto the gelas a positive control. The proteins were transferred to a nitrocellulosemembrane overnight at a 12 mA constant current in transfer buffer (39 mMglycine, 48 mM Tris-HCl, 20% methanol) at 4° C.

[0122] Following protein transfer to the nitrocellulose membrane, theblots were immediately placed into blocking buffer (5% non-fat dry milk,10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and incubated for30′ at room temperature. The blots were then individually incubatedovernight with mAbs to IRF-1, IRF-2, PKR, cytokeratin-18 (SANTA CRUZBIOTECHNOLOGY Inc, Santa Cruz, Calif.), iNOS (TRANSDUCTION LABORATORIES,Lexington, Ky.) and phospho-eIF-2a (CELL SIGNALING, Beverly, Mass.) at4° C. Blots were washed three times in washing buffer (10 mM Tris-HCl,pH 7.5, 100 mM NaCl, 0.1% Tween 20) and were subsequently incubated withanti-mouse IgG HRP conjugate (BOEHRINGER MANNHEIM, Indianapolis, Ind.)(1:5000) for 30′ at room temperature. The blots were again washed inwashing buffer and developed by the addition of ECL chemiluminescentdetection reagents (0.125 ml/cm²) according to the manufacturer'sinstructions (AMERSHAM LIFE SCIENCES, Arlington Heights, Ill.). Theblots were wrapped in saran wrap and exposed to Kodak X-OMAT AR films(EASTMAN KODAK, Rochester, N.Y.).

[0123] Nitrite Assay. Nitrite, a stable breakdown product of NO inphysiological systems, was assayed using the Griess reaction (23). Cellculture supernatants (100 μL) were added in triplicates to 1001L ofGriess reagent (sulfanilamide 1%, naphthylethylenediaminedihydrochloride 0.1%, phosphoric acid 2.5%) using 96-well plates (SIGMA,St. Louis, Mo.). After incubation at room temperature for 10 min,absorbance at 550 nm was measured. A doubling dilution of a 50 μM sodiumnitrite solution was used to generate a standard curve. The lower limitof the standard curve was 0.25 μM.

[0124] Northern Analysis. Northern blot analysis was performed toexamine the mRNA expression profile of IFN-γ-induced genes. Totalcellular RNA was isolated from cells using TRIZOL reagent (LifeTechnologies, Gaithersburg, Md.) following the manufacturer'sinstructions. Probes for northern hybridization were prepared by RT-PCRusing gene specific primers for IRF-1 (nucleotides 7-359), 2-5 AS p40(nucleotides 2-492), 2-5 AS p⁶⁹ (nucleotides 21-503), RSV G (nucleotides4688-5584), RSV F (nucleotides 5661-7385) and glyceraldehyde 3 phosphatedehydrogenase (GAPDH) (nucleotides 1-360). The PCR products wereconfirmed by sequencing. The probes were labeled using BrightStarPsoralen-Biotin labeling kit (AMBION, Austin, Tex.) followingmanufacturer's protocol. 10 mg of total RNA was size fractionated on 1%formaldehyde agarose gel, and transferred to nylon membranes (HYBOND N+,AMERSHAM, Piscataway, N.J.) using standard protocol (24) andcross-linked by UV irradiation (UV STRATALINKER 1800, STRATAGENE, SanDiego, Calif.). Hybridization was carried out at 420C overnight with 2-4pM labeled probe and UltraHyb hybridization solution (AMBION, Austin,Tex.).

[0125] The blots were washed twice with 2×SSC, 0.1% SDS for 5 minuteseach and two more washes with 0.1×SSC, 0.1% SDS for 15 minutes each at420C. The blots were processed for detection using the BRIGHTSTARBIODETECT Kit (AMBION, Austin, Tex.) following manufacturer's protocol.The blots were exposed to KODAK X-OMAT AR films (EASTMAN KODAK,Rochester, N.Y.) for 1-15 minutes. The bands were quantified by usingAdvanced Quantifier software (BIOIMAGE, Ann Arbor, Mich.) and thesignals were normalized with the respective GAPDH signal.

[0126] Antisense Blocking of 2′-5′ Oligoadenylate Synthetase.Phosphorothioate antisense oligonucleotides (ODNs) were designed againstp40 and p69 subunits of 2′-5′ oligoadenylate synthetase. The sequencesof antisense ODNs are as follows: p40 subunit, 5′-TTT CTG AGA TCC ATCATT GA-3′ (SEQ ID NO: 17) and p69 subunit, 5′-TCC CCA TTT CCC ATTGC-3′(SEQ ID NO: 18). The control ODN sequences 5′-GTC TAT GAA TAC TTTCCT AG-3′ (SEQ ID NO: 19) and 5′-CAC CTC TAT CTC TCT CG-3′ (SEQ ID NO:20) are a scramble of the antisense sequence to p40 and p69 isomers,respectively, i.e., identical in base composition. HEp-2 cells weretreated with 1000 U/ml of IFN-γ protein for 20 hours. At the same timeequimolar mixture of antisense ODNs to both the isoforms of 2-5 AS ortheir scrambled mismatch ODNs were added at concentrations 0, 3, 30 and90 nM. Cells were infected with RSV at 20 h post-IFN-γ-treatment, asdescribed earlier. After 1 h of virus adsorption, cultures weresupplemented with complete medium, which contained 1000 U/ml of IFN-γand respective concentrations of ODNs, and incubated for 72 hrs. ODNswere supplemented every 8 hours. At 72 h pi, cells were washed threetimes with cold PBS, pH 7.4, harvested and the clear cell homogenate wasused for plaque assay. 2-5 AS Assay. A 2-5 Assay was done following themethod described previously (Ghosh et al, J. Biol. Chem.272:15452-15458, 1997). Briefly, 20 μl of reaction mixture containingthe cell homogenate, 20 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate,2.5 mM dithiothreitol, 5 mM ATP, 5 μCi of [a-32P]ATP, and 50 μg/mlpoly(I).poly(C) was incubated for 3 h at 30° C. The reaction was stoppedby boiling for 3 min and centrifuged, and was incubated for 3 h at 37°C. with 3 μl of I unit/μl calf intestine alkaline phosphatase to convertthe unreacted [a-32P]ATP to inorganic phosphate. Two μl of the samplewere then spotted on a polyethyleneimine-cellulose thin layerchromatography plate and resolved in 750 mM KH2PO4, pH 3.5. The 2-5Aformed was then quantified by using Advanced Quantifier software(BIOIMAGE, Ann Arbor, Mich.) and expressed as arbitrary units.

[0127] Generation of Stable Cell Line Overexpressing Rnase L Inhibitor.Human RLI cDNA was amplified as KpnI-BamHI cassette and cloned inpcDNA3.1 (INVITROGEN, Carlsbad, Calif.) by the standard procedure(Sambrook et al., Molecular Cloning: A Laboratory Manual, and ed., ColdSpring Harbor Laboratory, NY, 1989). HEp-2 cells were transfected with 5mg of pcDNA3.1-RLI using lipofectine (LIFE TECHNOLOGIES, Gaithersburg,Md.). The empty pcDNA3.1 vector was used as a control. Stabletransfectants were selected by culturing the cells in the presence ofG418 (LIFE TECHNOLOGIES, Gaithersburg, Md.). Individual clones wereisolated and analyzed for the expression of RLI mRNA. The clone thatexpressed RLI at the highest level and had a normal morphology andgrowth pattern was selected and named RLI-14.

[0128] RNAse L Assay. An. RNAse L assay. was done by ribosomal RNAcleavage assay (Player et al., Methods, 15:243-253, 1998). Briefly,cells were harvested in NP-40 lysis buffer (10 mM HEPES, pH 7.5, 90 mMKCl, 1 mM magnesium acetate, 0.5% (v/v) NP-40, 2 mM 2-mercaptoethanol,100 mg/ml leupeptin) and S10 lysate was prepared and protein content wasestimated using the BCA (bicinchoninic acid) assay (Pierce, Rockford,Ill.). Ribosomal RNA cleavage by RNAse L was assayed in a 20 ml reactioncontaining 200 mg protein, 2 ml of 10× cleavage buffer (100 mM HEPES, pH7.5, 1 mM KCl, 50 mM magnesium acetate, 10 mM ATP, 0.14 M2-mercaptoethanol), 100 nM 2′-5′A and incubated at 300C for 2 h. RNA wasisolated from the reaction using TRIZOL reagent (LIFE TECHNOLOGIES,Gaithersburg, Md.) following the manufacturer's instructions. 2 mg ofRNA was separated on agarose gel electrophoresis and the rRNA cleavageproducts were compared.

[0129] Animals. Female 6-8 weeks old wild type and STAT4^(−/−) BALB/cmice from Jackson Laboratory (Bar Harbor, Me.) were maintained inpathogen free conditions at the animal center at USF College ofMedicine. All procedures were reviewed and approved by the committee onanimal research at the University of South Florida College of Medicine.

[0130] Cloning and recombination of adenoviral vectors. Murine 25AS(p40)cDNA was cloned into adenoviral transfer vector pShuttle-CMV(STRATAGENE, CA) at KpnI and XhoI sites. The left and right arms ofpShuttle-CMV vector contains AdS nucleotides 34,931-35,935 and3,534-5,790, which mediate homologous recombination with pAdEasy-1vector in E. coli, plus inverted terminal repeat (ITR) and packagingsignal sequences (nucleotides 1-480 of AdS) required for viralproduction in mammalian cells. pAdEasy-1 adenoviral plasmid (STRATAGENE,CA) contains all Ad5 sequences except nucleotides 1-3,533 (encompassingthe El gene) and nucleotides 28,130-30,820 (encompassing E3).

[0131] For generation of recombinant adenovirus plasmid,pShuttle-CMV-p40/LacZ plasmids were linearized with PmeI andco-transformed with pAdEasy-1 plasmid into recombination proficientBJ5183 cells. The recombination was confirmed by PacI digestion. Therecombined clones were re-transformed into DH5α cells for large-scaleplasmid purification.

[0132] Generation and purification of recombinant adenovirus. HEK293cells, which produce the deleted E1 genes in trans, were transfectedwith 4 μg of PacI digested recombinant adenovirus plasmid DNA withlipofectin (LIFE TECHNOLOGIES, MD). Cells were harvested 7-10 dayspost-transfection, resuspended in PBS and recombinant virus wascollected by 3-4 freeze-thaw cycles. The recombinant virus expressingmurine p40 and LacZ were termed Ad-p40 and Ad-LacZ, respectively. Theviruses were amplified by infecting fresh HEK-293 cells. Viruses werefurther purified by CsCl banding (Becker et al., Methods Cell Biol., 43Pt. A: 161-189, 1994). The viral band was extracted and CsCl was removedby passing through Centricon-100 columns (MILLIPORE, Mass.).

[0133] Quantitation of RSV titers in lung. To quantify RSV titers in themouse lung whole lungs were first weighed and placed immediately in EMEMmedia supplemented with 10% FBS. Lungs were homogenized, centrifuged at10,000 RPM for 10 minutes at 4° C., the clear supernatants were used forplaque assays by shell vial technique (Kumar et al., 2002).

[0134] Pulmonary Function. To evaluate the pulmonary function invaccinated and control groups, mice were administered IGT, as describedearlier. Three days later, airway responsiveness was assessednon-invasively in conscious, unrestrained mice with a whole bodyplethysmograph (BUXCO ELECTRONICS, Troy, N.Y.), as previously described(Matsuse et al., J. Immunol. 164:6583-6592, 2000). With this system, thevolume changes that occur during a normal respiratory cycle are recordedas the pressure difference between an animal-containing chamber and areference chamber. The resulting signal is used to calculate respiratoryfrequency, minute volume, tidal volume, and enhanced pause (Penh). Penhwas used as the measure of bronchoconstriction and was calculated fromthe formula: Penh=pause×(peak expiratory pressure/peak inspiratorypressure), where pause is the ratio of time required to exhale the last30% of tidal volume relative to the total time of expiration. Mice wereplaced in the plethysmograph and the chamber was equilibrated for 10min. They were exposed to aerosolized PBS (to establish baseline)followed by incremental doses (6, 12.5, 25, and 50 mg/ml) ofmethacholine (SIGMA CHEMICALS, St. Louis, Mo.). Each dose ofmethacholine was aerosolized for 5 min, and respiratory measurementswere recorded for 5 min afterward. During the recording period, anaverage of each variable was derived from every 30 breaths (or 30 s,whichever occurred first). The maximum Penh value after each dose asused to measure the extent of bronchoconstriction.

[0135] Bronchoalveolar lavage (BAL) and histology of the lung.Bronchoalveolar lavage were performed on Ad-p40 administered and controlmice, as described before (Kumar et al., 1999). Histological stainingand a semiquantitative analysis of airway inflammation from the lungs ofp40 treated and control groups of mice were performed, as describedearlier (Kumar et al., 1999). Lung inflammation was assessed afterstaining the sections with hematoxylin and eosin (HE). The entire lungsection was reviewed, and pathological changes were evaluated forepithelial damage, peribronchovascular cell infiltrate, andinterstitial-alveolar cell infiltrate for the mononuclear cells andpolmorphonuclear cells.

[0136] Statistical Analysis. Experiments were repeated 2 to 4 times foreach experiment as indicated. Statistical significance was analyzedusing paired two-tailed student's t-test. Differences were consideredstatistically significant when the p-value was less than 0.05.

Example 1 IFN-γ Attenuates RSV Infection in Human Epithelial Cells

[0137] To examine the effect of IFN-γ on RSV infection, HEp-2 cells werepre-incubated for 4-20 h with different concentrations of IFN-γ andsubsequently infected with RSV. Respective concentrations of IFN-γ wereadded back to the cells in complete medium after the removal of viralinoculum. Cells were harvested at 72 h p.i., and viral titer wasdetermined by plaque assay. RSV replication was inhibited significantlywith the addition of various concentrations of IFN-Y to both cell linesprior to RSV infection (FIGS. 1A and B). A 97% inhibition of replicationwas observed in HEp-2 and A549 cells, at 1000 U/ml of IFN-γ added at 20h pre-infection. Cells treated with IFN-γ 4 h pre-infection also showedsignificant reduction (p<0.01) in RSV titer (50% reduction). Asignificant decrease (p<0.01) in RSV titer (39% reduction) was observedin A549 cells, which were not treated with IFN-γ before infection, butwere only treated at 1 h post infection (FIG. 1C). To rule out thepossibility that the reduction in RSV titers could be due tocytotoxicity of IFN-γ, a MTT cytotoxicity assay was performed. Theresults indicate that the cells were metabolically as viable as theuntreated control cells when treated with the highest concentrations ofIFN-γ (1,000 U/ml; FIG. 1D). Thus, IFN-γ did not exhibit any cytotoxicor growth inhibitory effect on these cells. These results suggest thatthe treatment of cells with soluble IFN-γ results in a significantdecrease in RSV infection in epithelial cells.

Example 2 IFN-γ Induces IRF-1 Protein Expression

[0138] ISGs implicated in the antiviral activity of IFNs include IRFs,double stranded RNA activated protein kinase (PKR) and inducible nitricoxide synthase (iNOS). To identify the ISGs in these cells potentiallyinvolved in protection against RSV infection, proteins were analyzedfrom cells at various time points post treatment with IFN-γ (1000 U/ml).A western blot analysis was per-formed using specific antibodies toIRF-1, IRF-2 and PKR (FIG. 2A). There was increased expression ofIRF-1-but no change in the expression of IRF-2 following IFN-γ addition.Expression of IRF-1 increased after 30′ of IFN-γ addition. Theexpression of PKR decreased gradually over time (FIG. 2B) and no changein the expression of phospho-eIF-2a was observed following IFN-γaddition. Cytokeratin-18 was used as an internal control, the expressionof which did not change with the addition of IFN-γ. To examine if IFN-γinduced iNOS plays a role in antiviral action, iNOS expression wasexamined by western blotting (FIG. 2B). The expression of iNOS proteincould not be detected before and after IFN-γ addition. Murine macrophagecell lysate containing iNOS was used as a positive control, which didnot bind to the cytokeratin-18 antibody used as internal control. Torule out completely the involvement of iNOS in the antiviral effect ofIFN-γ, the level of nitric oxide (NO) was examined in the culturesupernatant of both HEp-2 and A549 cells before and after the additionof IFN-γ at various time points. No detectable level of NO (lowestconcentration of standard was 0.25 mM) was observed in both cell linesat any time point, i.e., before or after IFN-γ addition in both celllines. A similar expression pattern was observed for IRF1, IRF2, PKR andiNOS in A549 cells. These results indicate that IFN-γ up-regulates IRF-1in these cells and neither PKR nor iNOS play any role in the antiviralactivity of IFN-γ against RSV infection in human epithelial cell lines.

Example 3 Exogenous IFN-γ Upregulates mRNA Synthesis Of IRF-1 and 2-5 AS

[0139] IRF-1 has been reported to play a role in antiviral activity viathe induction and activation of 2-5 AS (Reis et al., EMBO J. 11:185-193,1992). Northern analysis was performed using gene specific probes forIRF-1 and the p40 and p69 isoforms of 2-5 AS (FIG. 3). The IRF-1 mRNAwas induced at 30 min after addition of IFN-γ and continued to increasegradually thereafter until 48 h post exposure. The induction of the p40and p69 isoforms of 2-5 AS was observed starting at 4 h and peaked at 24h post exposure. The p40 probe hybridized to two transcripts of 1.8 and1.6 kbp. Similarly, the p69 probe hybridized to four expectedtranscripts of 5.7, 4.5, 3.7 and 3.2 kbp of which 5.7 kbp was the majortranscript. These results suggest that IFN-γ induces IRF-1, which inturn, up regulates 2-5 AS, suggesting that the latter may be involved inthe anti-RSV mechanism of IFN-γ.

Example 4 2-5 As Antisense Oligonucleotides Abrogate the AntiviralEffect Of IFN-γ in HEp-2 Cells

[0140] To investigate whether IFN-induced anti-RSV activity is mediatedby 2-5 AS, IFN-γ exposed (1000 U/ml at 20 h pre-infection) HEp-2 cellswere treated with equimolar mixture of antisense oligonucleotides (ODNs)to both p40 and p69 isoforms of 2-5 AS. Scrambled mismatch of theantisense ODN sequence to p40 and p69 at the same concentration wereused as control. RSV infection was barely detectable in cells eithertreated with IFN-γ alone or with cells treated with IFN-γ and controlODNs but not in those treated with IFN-γ and antisense ODNs, as shown inFIG. 4. Addition of antisense ODN significantly reverted (p<0.01) theantiviral effect of IFN-γ against RSV infection and this reversal wasdose-dependent and increased with increasing concentrations of antisenseODNs. As shown in FIG. 4, 2-5 AS activity was reduced in a dosedependent manner in the cells treated with antisense ODN to 2-5 AS butnot control ODN. These results indicate that the addition of antisenseODNs to 2-5 AS to IFN-γ-treated cells reduced 2-5 AS activity in thesecells and in turn the antiviral effect of IFN-γ.

Example 5 Overexpression of Rnase L Inhibitor (RLI) Does Not Alter TheIFN-γ Responses in Hep-2 Cells

[0141] In addition to RNAse L, RNAse L inhibitor (RLI) has beenimplicated in the antiviral effect of IFN-γ. To determine the role of2-5A/RNase L-mediated antiviral mechanism, a stable cell line expressingRLI, RLI-14, was established. A northern analysis of RNAs from RLI-14and HEp-2 using gene specific probe for RLI showed a major 3.5 kbtranscript and aminor 2.8 Kb transcript (FIGS. 5A and 5B). A seven-foldincrease in the major RLI transcript expression was observed in RLI-14cells when compared to HEp-2 cells. The analysis of IFN-γ inducedproteins in RLI-14 cell line by western blotting showed that IFN-γinduced expression of IRF-1, but not IRF-2, at 30 min post induction andIRF-1 expression continued to increase thereafter until 48 h (FIG. 5C)as in HEp-2 cells (FIG. 2A). Also, a time-specific decrease in PKRprotein concentration was observed after IFN-γ addition in the RLI-14cell line. The expression of cytokeratin-18, used as an internalcontrol, remained unchanged with IFN-γ addition. The level of mRNAexpression of IRF-1, p40 and p69 isoforms of 2-5 AS was observed bynorthern analysis, and the expression level showed a gradual increaseover time following IFN-Y stimulation (FIG. 5D) as in HEp-2 cells (FIG.3). These results suggest that overexpression of RLI does not change theexpression pattern of the IFN-γ-induced genes involved in antiviralactivity of these cells.

Example 6 Rnase L Inhibitor (RLI) Overexpression Decreases the AntiviralActivity of IFN-γ

[0142] To examine the effect of the overexpression of the RNase Linhibitor, both HEp-2 and RLI-14 cells were treated with IFN-γ at100-1000 U/ml at 20 h pre-infection and subsequently infected with RSV.IFN-γ was added back to the cells at respective concentrations followingRSV infection. HEp-2 cells treated with 100 and 1000 U/ml of IFN-γshowed significant inhibition (p<0.001) of RSV infection (72% and 97%reduction, respectively) when compared to untreated cells. In markedcontrast, RLI-14 cells showed significantly lower inhibition ofinfection (only 12% and 22% reduction, respectively) compared to HEp-2cells at respective concentrations of IFN-γ, as showed in FIG. 6. Inabsence of IFN-γ treatment, both cell lines exhibited identical RSVtiters upon infection. However, the viral titer significantly decreased(p<0.01) when the concentration of IFN-γ was increased from 100 U/ml to1000 U/ml in RLI-14 cells. This demonstrates that increase in IFN-γ ledto higher expression of 2-5 AS and in turn production of 2-5A, whichsubsequently bound to RNase L and increased the level of active RNase Lby releasing RNase L from its inactive complex. Reduction in virusreplication was inhibited in RLI-14 cells (%) when compared to HEp-2cells (%), as shown in FIG. 6. In order to examine whether the reductionin inhibition of RSV infection in RLI-14 cells was due to reduced RNAseL activity in these cells, RNAse L assay was done using ribosomal RNAcleavage assay. This reaction uses cell lysate as a source of bothsubstrate and enzyme, thus giving a comparison of the ribonucleaseactivity of RNAse L in different cell types. The results confirm thatribonuclease activity of RNAse L is indeed reduced in RLI-14 cells whencompared to HEp-2 cells as evident from the rRNA cleavage products, asshown in FIG. 6. Together, these results confirm the involvement of2-5A/RNase L in the antiviral effect of IFN-γ against RSV infection.

[0143] The finding that treatment of HEp-2 and A549 cells at 20 hpre-infection with as low as 100 U/ml of IFN-γ proteins inhibits RSVinfection and replication when compared to untreated cells, hassignificant therapeutic implications. HEp-2 and A549 cells treated with1000 U/ml of IFN-γ at 20 h pre-infection exhibited a 97% (30-31 fold inloglO PFU/ml) reduction in RSV titer. The RSV titer also decreased by39% (1.7 fold reduction in log 10 PFU/ml) in these cells, which were nottreated with IFN-γ prior to infection but were only treated immediatelyafter RSV infection. RSV is resistant to the antiviral effects of type-Iinterferons and human M×A. It has been reported that overexpression ofIFN-γ by gene transfer and by recombinant RSV attenuates RSV replicationin a mouse model of RSV infection. However, the mechanism of antiviralaction of IFN-γ against RSV is not known.

[0144] The elucidation of the mechanism underlying IFN-γ-mediatedresistance to RSV infection in human epithelial cells has been the mainfocus of this invention. The mechanism of antiviral action of IFN-γ iscomplex and may be unique for individual cell lines and viruses. Aprofile of ISGs, relevant to antiviral activity in these epithelialcells, establish that IFN-γ exposure results in induction of both themRNA and protein for IRF-1 but not IRF2. In non-induced cells the IRF-2protein functions as a repressor of ISGs. IFN-γ induction temporarilyremoves this repression and activates ISGs including IRF-1. IRF-1 andIRF-2 compete for the same cis acting recognition sequences but withopposite effects. Findings in these epithelial cells are consistent withthose found for human macrophages, where IFN-γ treatment does notenhance IRF-2 gene expression, despite strong upregulation of IRF-1 mRNAexpression. Two additional ISGs, PKR and iNOS proteins were examined fortheir role in IFN-γ induced antiviral activity. IFN-γ activates PKR,which in turn phosphorylates and inactivates eukaryotic initiationfactor-2a (eIF-2a) and leads to restriction of cellular as well as viralprotein synthesis. The iNOS is also known to mediate antiviral propertyof IFN-γ. However, a time specific decrease in PKR expression and nochange in phosphorylation of eIF-2a and the lack of detectable levels ofiNOS protein or NO in IFN-γ-stimulated HEp-2 and A549 cells indicatethat neither PKR and phospho-eIF-2a nor iNOS play any role in IFN-γmediated inhibition of RSV infection in these cells.

[0145] To further dissect the mechanism of IFN-γ mediated anti-RSVactivity in HEp-2 and A549 cells, IRF-1 induced expression of 2-5 AS wasexamined. Of the four isoforms (p40, p46, p69, and p110) of 2-5 ASdetected in human cells to date, the expression pattern of the p40 andp69 isoforms following IFN-γ stimulation was examined in this studybecause of the following. The p40 and p46 isoforms of 2-5 AS, which aredependent on dsRNA for activation, are derived from the same gene bydifferential splicing between the fifth and an additional sixth exon ofthis gene and are thus identical for the first 346 residues, except fortheir C-terminal ends. Of the two high molecular weight isoforms, p69,but not p100, requires dsRNA for activation. The expression of 2-5 ASp40 and p69 are induced by IFN-γ in these cells at 4 h and peaks at 24 hpost IFN-γ addition. Therefore, the antiviral effect of IFN-γ in thesecells is observed when the cells are treated with IFN-γ at 4 hpre-infection and is highest when treated at 20 h pre-infection as thelevel of 2-5 AS is at peak at that time. These data suggest that theantiviral mechanism of IFN-γ against RSV infection is mediated by theactivation of IRF-1, which in turn activates the 2-5 AS system. Adose-dependent abrogation of 2′-5′ AS activity and in turn the anti-RSVeffect of IFN-γ by the addition of an equimolar mixture of antisenseODNs to p40 and p69, but not by the scrambled mismatch ODNs, provideevidence supporting the role of 2-5 AS in the antiviral mechanism ofIFN-γ against RSV infection.

[0146] 2-5 AS induces 2-5A, which binds to and activate RNase L, whichcleaves double stranded RNA 3′ of UpN residues. The levels of RNase Lare increased in growth-arrested cells and following IFN-γ treatment;however, its biological activity is thought to be controlled at thelevel of enzymatic activation rather than through regulation of itstranscription and translation. Increasing endogenous levels of 2-5Aleads to enhanced RNase L activity, which suggests that intracellularlevels of 2-5A are rate limiting in the activation of RNase L, whereascellular levels of RNase L are sufficient for maximal biologicalactivity. Furthermore, RNase L remains in an inactive form in the cellsbeing bound to an inhibitor, RLI, which codes for a 68 kDa protein whosemRNA is not regulated by IFN-γ. RLI induces neither 2-5A degradation norreversible modification of RNase L when expressed in a reticulocytelysate, but antagonizes the binding of 2-5A to RNase L, thus, itsnuclease activity, since 2-5A binding is a prerequisite to RNase Ldimerization and activation.

[0147] RLI-14, a stable cell line overexpressing RLI, was establishedfrom HEp-2 cells and characterized to determine precisely theinvolvement of RNase L in the antiviral mechanism of RSV infectedepithelial cells. The finding that RLI-14 was almost identical to theparent HEp-2 cells in its response to IFN-γ shows that RLIoverexpression does not alter the induction of ISGs in these cells(FIGS. 5A-D). Nonetheless, reduced RNAse L activity and antiviralactivity of IFN-γ in RLI-14 cells (FIG. 6), confirmed that the RNase Lactivity is indeed critical to the antiviral effect of IFN-γ and is onlypartly controlled by the elevated levels of 2-5 AS in these cellsfollowing IFN-γ treatment. The reduction in antiviral effect of IFN-γ inthese cells was dependent on the dose of IFN-γ, indicating that thelevel of 2-5A, which is regulated by IFN-γ and the level of RLI arecrucial in determining which pathway cells will follow. The importanceof the level of 2-5A was confirmed by preliminary findings which showedsignificant reduction in RSV infection when HEp-2 cells were treatedwith 100 U/ml of IFN-γ 20 h pre-infection and transfected with 1 mM 2-5A2 h pre-infection, when compared to the cells treated with 100 U/ml ofIFN-g alone. Similarly the importance of the level of RLI in theantiviral activity was reported for HIV, where RLI is induced duringHIV1 infection and down regulates the 2-5A/RNase L pathway in human Tcells.

[0148] In summary, these results demonstrate that IFN-γ inhibits RSVinfection of human epithelial cells. Specifically, in HEp-2 and A549epithelial cells, IFN-γ upregulates IRF-1, which in turn, induces 2-5AS. Further, the 2-5 AS generates 2-5A that activates RNase L, which isnormally found in the cytoplasm in inactive state bound to RLI. Thus,RNase L-mediated cleavage of viral RNA is governed by the ying-yangmechanism involving 2-5A and RLI. In a 2-5A-dominant state cells areprotected from RSV infection due to the activation of RNase L. Incontrast, an RLI-dominant condition attenuates the antiviral effect byinactivation of RNase L. Since, 2-5A and RLI are respectively, governedby IFN-γ-dependent and independent mechanisms, treatment with IFN-γ oroverexpression of 2-5 AS should provide an efficient means to redirectthe 2-5A:RLI ratio toward a shift in favor of 2-5A and achieve aprofound antiviral effect.

Example 7 2-5 AS Plasmid DNA Vaccination Attenuates RSV Infection andPathogenesis

[0149] As shown in FIG. 8A, 2-5 AS pDNA vaccine decreases lung RSVtiters. BALB/c mice (n=4) were intranasally administered with p2′-5′ AS(25 mg of DNA each time complexed with lipofectamine) or an equal amountof empty pVAX vector DNA 3 times in 2-day intervals. Mice were infectedwith RSV seven days after last DNA administration and were sacrificed onday 5 post-infection. BAL was performed and lungs were collected. RSVtiter was determined by plaque assay from the lung homogenate. Theresults show that 2-5 AS cDNA vaccination can significantly attenuatelung titers of RSV in infected mice.

[0150]FIG. 8B shows that reduction of viral titers is associated withreduction in MIP-1α. Expression level of MIP-1α was determined from lunghomogenate by ELISA. The results show that vaccination with 2-5 AS cDNAdecreases production of chemokine MIP-1α which is known to beproinflammatory in action.

[0151] In FIG. 8C, 2-5 AS overexpression increased the macrophagepopulation significantly compared to RSV infected mice. BAL celldifferential was performed and percentages of macrophage, lymphocytes,and neutrophils were determined. The results show that 2-5 AS does notalter the cellular composition of the lung. No significant changes areseen in lymphocytes and macrophages, however the percent of neutrophilsis increased in the lungs of mice treated with 2-5 AS cDNA. FIGS. 9A-9Cshow that 2-5 AS vaccination significantly decreased pulmonaryinflammation. Histological sections from lungs were stained withhematoxylin and eosin and representative photomicrographs are shown.

Example 8 Ad-2-5AS(p40) Decreases Lung RSV Titers

[0152] A reduction in virus titer is the gold standard by which theeffectiveness of an antiviral therapy is measured. Mice wereintranasally administered with 108 PFU/ml rAD-p40 and then infected withRSV 4 h later. Analysis of lung virus titers following acute, live RSVinfection at day 5 post infection show a significant (100-fold, P<0.01)reduction in RSV titers in Ad-p40 treated mice compared to controls(FIG. 10). These results indicate that the rAD-p40 treatment constitutesan effective prophylaxis against RSV infection.

Example 9 Ad-2-5AS(p40) Decreases AHR in Mice

[0153] The safety of an antiviral therapy especially can be measured bya decrease in RSV-induced AHR. To test whether Ad-p40 administrationreduces RSV-induced airway hyperreactivity, the % baseline enhancedpause (Penh) was measured in a group of mice treated with rAD-p40 priorto RSV infection and their AHR was compared with untreated RSV infectedgroup. Mice receiving Ad-p40 exhibited a similar response tomethacholine challenge when compared to uninfected PBS control group(FIG. 10). These results suggest that the Ad-p40 induced decrease in RSVinfection decreases AHR.

Example 10 Ad-2-5AS(p40) Normalizes Cellular Infiltration to the Lung

[0154] The inflammation in the lung due to RSV infection is dueinfiltration into the lung a large number of macrophages andlymphocytes. To determine whether treatment with rAD-p40 affectsmigration of these cells to the lung, mice administered with rAD-p40 andRSV infected were compared to RSV infected mice without treatment andnaive mice as control and to rAd-lacZ as control. Mice with p40 genetransfer and RSV infection show a BAL cell differential similar to thatof normal uninfected animals, lack of AHR compared to RSV-infectedanimals without p40 gene transfer and lack of the peribronchiolar andperivascular inflammation suggesting that intranasal p40 can potentiallybe an effective anti-viral approach in vivo for RSV infection.

Example 11 Ad-2-5AS(p40) Decreases RSV Infection-Induced PulmonaryInflammation

[0155] Lung inflammation was examined in different groups of mice. Micetreated with Ad-p40 and Ad-lacZ upon acute RSV infection exhibitrelatively less disruption of the epithelium and cellular infiltration.Representative pathological features reveal that group of mice receivingAd-p40 exhibit less epithelial damage, and reduced mononuclear cell(MNC) and polymorphonuclear cell (PMNC) infiltrates in the interstitialand peribronchovascular region, as compared to Ad-lacZcontrols (FIGS.12A-12H). These results suggest that the Ad-p40 protects mice from RSVinfection-induced pulmonary inflammation. These results suggest thatAd-p40 protects mice from RSV infection-induced pulmonary inflammation.

[0156] The finding that transient gene expression therapy cansubstantially reduce lung RSV titers by 2 logs (100-fold), inhibitRSV-infection induced AHR and make the lung resistant to inflammation byacute RSV infection is very significant. These results suggesttremendous therapeutic potential of this approach. The other members ofthis family include the measles virus, the sendai virus, parainfluenza1, 2, and 3, the mumps virus, the simian virus, and the newcastle virus.This finding is also important for other Paramyxoviruses, such asrotavirus that causes juvenile diarrhea in millions of childrenworldwide. Furthermore, beyond this family of viruses, the 2-5 AS/RNaseL cascade has been implicated in the anti-viral activity of picomaviruses, (Hassel, B A et al. Embo J, 1993, 12(8):3297-304; Benavente, Jet al. J Virol. 1984, 51(3):866-71; Goswami, B B and Sharma, O K. J.Biol Chem, 1984, 259(3):1371-4; Nilsen, T W et al. Mol Cell Biol, 1983,3(1):64-9), vaccinia virus (Maitra, R K and Silverman, R H. J. Virol,1998, 72(2):1146-52; Banerjee, R et al. Virology, 1990, 179(1):410-5),reovirus (Kumar, R et al. J. Virol, 1988, 62(9):3175-81), HIV (Saito, Het al. Keio J Med, 1996, 45(3):161-7; 45), EMCV (Glezen, W P et al. Am JDis Child, 1986, 140(6):543-6), Hepatitis B and C virus (Groothuis, J Ret al. Pediatrics, 1988, 82(2):199-203; Nelson W E, Behrman R E,Kliegman R. Nelson Textbook of Pediatrics. 15 ed. Philadelphia).

[0157] Moreover, besides human disease, this finding may haveimplications, for RSV of cattle (BRSV), sheep, and goats. If 2-5ASmediated approach is successful, the mortality and morbidity due to RSVinfection can be reduced. Also, RSV has been linked with the developmentof asthma, and hence, prevention or successful treatment of RSV isexpected to decrease the incidence of asthma and fatal exacerbation ofasthma due to RSV. Adults infected with RSV miss work for an average of7-9 days as opposed those with flu who miss an average of 6-7 days.Therapeutic treatment can reduce the number of absences from the work,which exceeds the flu infection. Also, prophylaxis prior to and duringviral season and treatment immediately after infection can lead to asubstantial decrease in hospitalization and emergency visits due to RSVinfection.

[0158] RSV is one of the important viral respiratory pathogen thatproduces an annual epidemic of respiratory illness. In children, commondiseases associated with RSV infection primarily include interstitiallung diseases, such as bronchiolitis, and asthma. RSV is estimated tocause 85% of the cases of acute bronchiolitis that affects infants andyoung children (Shay, D K et al. Jama, 1999, 282(15):1440-6). Somechildren may become infected during three or four successive RSVseasons. Each year, RSV is responsible for up to an estimated 125,000pediatric hospitalizations, with a mortality rate of approximately 2%(Heilman, Calif. J Infect Dis 1990, 161(3):402-6; Shay, DK et al. JInfect Dis, 2001, 183(1):16-22; Altman, Calif. et al. Pediatr Cardiol,2000, 21(5):433-8; Simoes, E A. Lancet, 1999, 354(9181):847-52; Falsey,Ark. et al. J Infect Dis, 1995, 172(2):389-94). Among hospitalizedinfants with chronic lung and heart disease, the RSV mortality rate maybe as high as 5%. Up to half of all pediatric admissions forbronchiolitis and one-quarter of admissions for pneumonia are due to RSV(La Via et al., Clin. Pediatr. (Phila), 32(8):450-454, 1993). RSV is amajor risk factor for a number of other health conditions, such asimmuno-deficiency, cardiac arrhythmia, congenital heart disease, andunusual atrial tachycardia (Donnerstein et al., J. Pediatr.125(1):23-28, 1994).

[0159] Emerging evidence also suggests that RSV is an important pathogenin profusely healthy adults as well (Hall et al., Clin. Infect. Dis.33(6):792-796, 2001). In a study of 15 adults who were challenged by RSVafter a natural infection, 50% were reinfected after two months; by 26months 73% were reinfected (Fixler, D E. et al. Pediatr Cardiol, 1996,17(3):163-8). RSV infection is also clinically important in previouslyhealthy working adults (Hogg, J C. et al. American Journal ofRespiratory & Critical Care Medicine, 1999, 160(5):S26-S28). In thisstudy, of a total of 2960 18-60 year-old adults studied during 1975 to1995, 211 (7%) acquired RSV infection; 84% of infections weresymptomatic −74% upper respiratory tract infection, 26% lowerrespiratory tract infection and 40% were febrile. RSV is a major riskfactor for the development and/or exacerbation of asthma and chronicobstructive pulmonary disorder (COPD), and about 30 million of Americanssuffers from asthma and COPD.

[0160] The prevalence of bronchiolitis in infants as well as asthma andCOPD has increased throughout the world, including in the United States,over the past two decades. The rates of death from asthma have increasedfrom 0.8 per 100,000 in 1977-78 to 2.0 per 100,000 in 1991, and theserates have increased in almost all age groups in the United States (Sly,R M. Ann Allergy, 1994, 73(3):259-68). Asthma is the most common causeof the admission of children to the hospital, and it is the most commonchronic illness causing absence from school and work in North America(Nelson, RP, Jr., et al. J Allergy Clin Immunol, 1996, 98(2):258-63).The total costs of illnesses related to asthma in 1990 were 6.2 billion,a 53% increase in direct medical expenditures and a 23% increase inindirect costs since 1985 (Weiss, K B et al. N Engl J Med, 1992,326(13):862-6). The total estimated cost in 1995 for the treatment ofallergic diseases, asthma, chronic sinusitis, otitis media, and nasalpolyps, was about $10 billion (Baraniuk, J N. J Resp Dis, 1996, 17(S11)). Together, these diseases lead to a significant reduction in thequality of life and a tremendous economic loss.

[0161] Finally, although studies with 2-5AS (p40) in the adenovirussystem provides the “proof of concept” for the anti-RSV activity, othervirus vectors, including adeno-associated vectors (Zhao, N et al. MolBiotechnol, 2001, 19(3):229-37; Monahan, P E et al. Mol Med Today, 2000,6(11):433-40; Senior, K. Lancet, 2002, 359(9313):1216) can be used toexpress this p-40 or other 2-5AS gene(s) for the antiviral activity.

Example 12 Gene Therapy

[0162] In the therapeutic and prophylactic methods of the presentinvention, the nucleotide sequence encoding 2-5 AS, or a catalyticallyactive fragment thereof, can be administered to a patient in variousways. It should be noted that the.vaccine can be administered as thecompound or as pharmaceutically acceptable salt and can be administeredalone or as an active ingredient in combination with pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles. In those cases inwhich the RNA virus is a virus that infects the patient's respiratorysystem, the compounds can be administered intranasally, bronchially, viainhalation pathways, for example. The patient being treated is awarm-blooded animal and, in particular, mammals including man. Thepharmaceutically acceptable carriers, diluents, adjuvants and vehiclesas well as implant carriers generally refer to inert, non-toxic solid orliquid fillers, diluents or encapsulating material not reacting with theactive ingredients of the present invention.

[0163] It is noted that humans are treated generally longer than themice exemplified herein, which treatment has a length proportional tothe length of the disease process and drug effectiveness. The doses maybe single doses or multiple doses over a period of several days, butsingle doses are preferred.

[0164] The carrier for gene therapy can be a solvent or dispersingmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils.

[0165] Proper fluidity, when desired, can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,olive oil, soybean oil, corn oil, sunflower oil, or peanut oil andesters, such as isopropyl myristate, may also be used as solvent systemsfor compound compositions. Additionally, various additives that enhancethe stability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

[0166] Examples of delivery systems useful in the present inventioninclude, but are not limited to: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other delivery systems andmodules are well known to those skilled in the art.

[0167] A pharmacological formulation of the nucleotide sequence utilizedin the present invention can be administered orally to the patient.Conventional methods such as administering the compounds in tablets,suspensions, solutions, emulsions, capsules, powders, syrups and thelike are usable. Known techniques which deliver the vaccine orally orintravenously and retain the biological activity are preferred.

[0168] In one embodiment, the nucleotide sequence can be administeredinitially by nasal infection to increase the local levels of 2-5 ASenzymatic activity. The patient's 2-5 AS activity levels are thenmaintained by an oral dosage form, although other forms ofadministration, dependent upon the patient's condition and as indicatedabove, can be used. The quantity of vaccine to be administered will varyfor the patient being treated and will vary from about 100 ng/kg of bodyweight to 100 mg/kg of body weight per day and preferably will be from10 mg/kg to 10 mg/kg per day.

[0169] As indicated above, standard molecular biology techniques knownin the art and not specifically described can be generally followed asin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (1989), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley& Sons, New York (1988), and in Watson et al., Recombinant DNA,Scientific American Books, New York and in Birren et al. (eds) GenomeAnalysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring HarborLaboratory Press, New York (1998) and methodology as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057, thecontents of which are incorporated herein by reference in theirentirety. Polymerase chain reaction (PCR) can be carried out generallyas in PCR Protocols: A Guide To Methods And Applications, AcademicPress, San Diego, Calif. (1990). In-situ (In-cell) PCR in combinationwith Flow Cytometry can be used for detection of cells containingspecific DNA and mRNA sequences (Testoni et al., 1996, Blood 87:3822).

[0170] As used herein, the term “gene therapy” refers to the transfer ofgenetic material (e.g., DNA or RNA) of interest into a host to treat orprevent a genetic or acquired disease or condition phenotype. Thegenetic material of interest encodes a product (e.g., a protein,polypeptide, peptide or functional RNA) whose production in vivo isdesired. For example, in addition to the nucleotide encoding 2-5 AS, ora catalytically active fragment thereof, the genetic material ofinterest can encode a hormone, receptor, or other enzyme, polypeptide orpeptide of therapeutic value. For a review see, in general, the text“Gene Therapy” (Advances in Pharmacology 40, Academic Press, 1997).

[0171] Two basic approaches to gene therapy have evolved: (1) ex vivoand (2) in vivo gene therapy. In ex vivo gene therapy, cells are removedfrom a patient, and while being cultured are treated in vitro.Generally, a functional replacement gene is introduced into the cell viaan appropriate gene delivery vehicle/method (transfection, transduction,homologous recombination, etc.) and an expression system as needed andthen the genetically modified cells are expanded in culture and returnedto the host/patient. These genetically reimplanted cells produce thetransfected gene product in situ. Alternatively, a xenogenic orallogeneic donor's cells can be genetically modified with the nucleotidesequence in vitro and subsequently administered to the patient.

[0172] In in vivo gene therapy, target cells are not removed from thepatient; rather, the gene to be transferred is introduced into the cellsof the recipient organism in situ, that is within the recipient.Alternatively, if the host gene is defective, the gene is repaired insitu. These genetically modified cells produce the transfected geneproduct in situ.

[0173] The gene expression vehicle is capable of delivery/transfer ofheterologous nucleic acids into a host cell. As indicated previously,the expression vehicle may include elements to control targeting,expression and transcription of the nucleotide sequence in a cellselective or tissue-specific manner, as is known in the art. It shouldbe noted that often the 5′UTR and/or 3′UTR of the gene may be replacedby the 5′UTR and/or 3′UTR of the expression vehicle. Therefore as usedherein the expression vehicle may, as needed, not include the 5′UTRand/or 3′UTR and only include the specific amino acid coding region.

[0174] The expression vehicle can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that may be required to obtain necessary transcription levelscan optionally be included. Enhancers are generally any non-translatedDNA sequence which works contiguously with the coding sequence (in cis)to change the basal transcription level dictated by the promoter. Theexpression vehicle can also include a selection gene as described hereinbelow.

[0175] Vectors can be introduced into cells or tissues by any one of avariety of known methods within the art. Such methods can be foundgenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992); inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press,Ann Arbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al(1986) and include, for example, stable or transient transfection,lipofection, electroporation and infection with recombinant viralvectors. In addition, see U.S. Pat. Nos. 4,866,042 for vectors involvingthe central nervous system and also U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

[0176] Introduction of nucleic acids by infection offers severaladvantages over the other listed methods. Higher efficiency can beobtained due to their infectious nature. Moreover, viruses are veryspecialized and typically infect and propagate in specific cell types.Thus, their natural specificity can be used to target the vectors tospecific cell types in vivo or within a tissue or mixed culture ofcells. Viral vectors can also be modified with specific receptors orligands to alter target specificity through receptor mediated events.

[0177] A specific example of a DNA viral vector for introducing andexpressing recombinant nucleotide sequences is the adenovirus derivedvector Adenop53TK. This vector expresses a herpes virus thymidine kinase(TK) gene for either positive or negative selection and an expressioncassette for desired recombinant sequences. This vector can be used toinfect cells that have an adenovirus receptor which includes mostcancers of epithelial origin as well as others. This vector as well asothers that exhibit similar desired functions can be used to treat amixed population of cells and can include, for example, an in vitro orex vivo culture of cells, a tissue or a human subject.

[0178] Additional features can be added to the vector to ensure itssafety and/or enhance its therapeutic efficacy. Such features include,for example, markers that can be used to negatively select against cellsinfected with the recombinant virus. An example of such a negativeselection marker is the TK gene described above that confers sensitivityto the antibiotic gancyclovir. Negative selection is therefore a meansby which infection can be controlled because it provides induciblesuicide through the addition of antibiotic. Such protection ensures thatif, for example, mutations arise that produce altered forms of the viralvector or recombinant sequence, cellular transformation will not occur.Features that limit expression to particular cell types or tissue typescan also be included. Such features include, for example, promoter andregulatory elements that are specific for the desired cell type ortissue type.

[0179] In addition, recombinant viral vectors are useful for in vivoexpression of a desired nucleic acid because they offer advantages suchas lateral infection and targeting specificity. Lateral infection isinherent in the life cycle of, for example, retrovirus and is theprocess by which a single infected cell produces many progeny virionsthat bud off and infect neighboring cells. The result is that a largearea becomes rapidly infected, most of which was not initially infectedby the original viral particles. This is in contrast to vertical-type ofinfection in which the infectious agent spreads only through daughterprogeny. Viral vectors can also be produced that are unable to spreadlaterally. This characteristic can be useful if the desired purpose isto introduce a specified gene into only a localized number of targetedcells.

[0180] As described above, viruses are very specialized infectiousagents that have evolved, in many cases, to elude host defensemechanisms. Typically, viruses infect and propagate in specific celltypes. The targeting specificity of viral vectors utilizes its naturalspecificity to specifically target predetermined cell types and therebyintroduce a recombinant gene into the infected cell. The vector to beused in the methods of the present invention will depend on desired thecell type or cell types to be targeted and will be known to thoseskilled in the art. For example, if RSV infection is to be inhibited(i.e., treated or prevented), then a vector specific for suchrespiratory mucosal epithelial cells would preferably be used.

[0181] Retroviral vectors can be constructed to function either asinfectious particles or to undergo only a single initial round ofinfection. In the former case, the genome of the virus is modified sothat it maintains all the necessary genes, regulatory sequences andpackaging signals to synthesize new viral proteins and RNA. Once thesemolecules are synthesized, the host cell packages the RNA into new viralparticles that are capable of undergoing further rounds of infection.The vector's genome is also engineered to encode and express the desiredrecombinant nucleotide sequence. In the case of non-infectious viralvectors, the vector genome is usually mutated to destroy the viralpackaging signal that is required to encapsulate the RNA into viralparticles. Without such a signal, any particles that are formed will notcontain a genome and therefore cannot proceed through subsequent roundsof infection. The specific type of vector will depend upon the intendedapplication. The actual vectors are also known and readily availablewithin the art or can be constructed by one skilled in the art usingwell-known methodology.

[0182] The recombinant vector can be administered in several ways. Ifviral vectors are used, for example, the procedure can take advantage oftheir target specificity and consequently, do not have to beadministered locally at the diseased site. However, local administrationcan provide a quicker and more effective treatment, administration canalso be performed by, for example, intravenous or subcutaneous injectioninto the subject. Injection of the viral vectors into a spinal fluid canalso be used as a mode of administration, especially in the case of RNAvirus infections of the central nervous system. Following injection, theviral vectors will circulate until they recognize host cells with theappropriate target specificity for infection.

[0183] An alternate mode of administration can be by direct inoculationlocally at the site of the disease or pathological condition or byinoculation into the vascular system supplying the site with nutrientsor into the spinal fluid. Local administration is advantageous becausethere is no dilution effect and, therefore, a smaller dose is requiredto achieve expression in a majority of the targeted cells. Additionally,local inoculation can alleviate the targeting requirement required withother forms of administration since a vector can be used that infectsall cells in the inoculated area. If expression is desired in only aspecific subset of cells within the inoculated area, then promoter andregulatory elements that are specific for the desired subset can be usedto accomplish this goal. Such non-targeting vectors can be, for example,viral vectors, viral genome, plasmids, phagemids and the like.Transfection vehicles such as liposomes and colloidal polymericparticles can also be used to introduce the non-viral vectors describedabove into recipient cells within the inoculated area. Such transfectionvehicles are known to those skilled within the art.

[0184] Direct DNA inoculations can be administered as a method ofvaccination. Plasmid DNAs encoding influenza virus hemagglutininglycoproteins have been tested for the ability to provide protectionagainst lethal influenza challenges. In immunization trials usinginoculations of purified DNA in saline, 67-95% of test mice and 25-63%of test chickens were protected against the lethal challenge. Goodprotection was achieved by intramuscular, intravenous and intradermalinjections. In mice, 95% protection was achieved by gene gun delivery of250-2500 times less DNA than the saline inoculations. Successful DNAvaccination by multiple routes of inoculation and the high efficiency ofgene-gun delivery highlight the potential of this promising new approachto immunization. Plasmid DNAs expressing influenza virus hemagglutininglycoproteins have been tested for their ability to raise protectiveimmunity against lethal influenza challenges of the same subtype. Intrials using two inoculations of from 50 to 300 micrograms of purifiedDNA in saline, 67-95% of test mice and 25-63% of test chickens have beenprotected against a lethal influenza challenge. Parenteral routes ofinoculation that achieved good protection included intramuscular andintravenous injections. Successful mucosal routes of vaccinationincluded DNA drops administered to the nares or trachea. By far, themost efficient DNA immunizations were achieved by using a gene gun todeliver DNA-coated gold beads to the epidermis. In mice, 95% protectionwas achieved by two immunizations with beads loaded with as little as0.4 micrograms of DNA. The breadth of routes supporting successful DNAimmunizations, coupled with the very small amounts of DNA required forgene-gun immunizations, highlight the potential of this remarkablysimple technique for the development of subunit vaccines. In contrast tothe DNA based antigen vaccines, the present invention provides thedevelopment of an intranasal gene transfer method using 2-5 AS, or acatalytically active fragment thereof, which can be used as aprophylaxis against multiple respiratory infections. In a preferredembodiment, the preventative and therapeutic method is used againstrespiratory RNA viral infection, most preferably against RSV.

[0185] All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

[0186] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 20 <210> SEQ ID NO 1<211> LENGTH: 1203 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1203) <223> OTHERINFORMATION: <300> PUBLICATION INFORMATION: <301> AUTHORS: Aissouni, Y.,et al. <302> TITLE: The cleavage/polyadenylation activity triggered by aU-rich motif sequence <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 277<305> ISSUE: 39 <306> PAGES: 35808-35814 <307> DATE: 2002 <308> DATABASEACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06<300> PUBLICATION INFORMATION: <301> AUTHORS: Behera, A.K., et al. <302>TITLE: 2′-5′ Oligoadenylate synthesis plays a critical role ininterferon-gamma inhibition <303> JOURNAL: J. Biol. Chem. <304> VOLUME:277 <305> ISSUE: 28 <306> PAGES: 25601-25608 <307> DATE: 2002 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Sarker, S.N.,et al. <302> TITLE: Identification of the substrate-binding sites of2′-5′- oligoadenylate synthetase <303> JOURNAL: J. Biol. Chem. <304>VOLUME: 277 <305> ISSUE: 27 <306> PAGES: 24321-24330 <307> DATE: 2002<308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRYDATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS:Hovnanian, A., et al. <302> TITLE: The human 2′,5′-oligoadenylatesynthetase locus is composed of three distinct genes <303> JOURNAL:Genomics <304> VOLUME: 52 <305> ISSUE: 3 <306> PAGES: 267-277 <307>DATE: 1998 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Renault, B., et al. <302> TITLE: A sequence-ready physical mapof a region of 12q24.1 <303> JOURNAL: Genomics <304> VOLUME: 45 <305>ISSUE: 2 <306> PAGES: 271-278 <307> DATE: 1997 <308> DATABASE ACCESSIONNUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300>PUBLICATION INFORMATION: <301> AUTHORS: Nechiporuk, T., et al. <302>TITLE: A high-resolution PAC and BAC map of the SCA2 region <303>JOURNAL: Genomics <304> VOLUME: 44 <305> ISSUE: 3 <306> PAGES: 321-329<307> DATE: 1997 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Wathelet, M.G., et al. <302> TITLE: Cloning and chromosomallocation of human genes inducible by type I interferon <303> JOURNAL:Somat. Cell Mol. Genet. <304> VOLUME: 14 <305> ISSUE: 5 <306> PAGES:415-426 <307> DATE: 1988 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Rutherford, M.N., et al. <302> TITLE: Interferon-inducedbinding of nuclear factors to promoter elements of the 2-5A synthetasegene <303> JOURNAL: EMBO J. <304> VOLUME: 7 <305> ISSUE: 3 <306> PAGES:751-759 <307> DATE: 1988 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Wathelet, M.G., et al. <302> TITLE: New inducers revealedby the promoter sequence analysis of two interferon-activated humangenes <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 169 <305> ISSUE: 2<306> PAGES: 313-321 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Benech, P., et al. <302> TITLE:Interferon-responsive regulatory elements in the promoter of the human2′,5′-oligo(A) synthetase gene <303> JOURNAL: Mol. Cell. Biol. <304>VOLUME: 7 <305> ISSUE: 12 <306> PAGES: 4498-4504 <307> DATE: 1987 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Hovanessian,A.G., et al. <302> TITLE: Identification of 69-kd and 100-kd forms of2-5A synthetase <303> JOURNAL: EMBO J. <304> VOLUME: 6 <305> ISSUE: 5<306> PAGES: 1273-1280 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Williams, B.R., et al. <302> TITLE:Interferon-regulated human 2-5A synthetase gene maps to chromosome <303>JOURNAL: Somat. Cell Mol. Genet. <304> VOLUME: 12 <305> ISSUE: 4 <306>PAGES: 403-408 <307> DATE: 1986 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Shiojiri, S., et al. <302> TITLE: Structureand expression of a cloned cDNA for human (2′-5′) oligoadenylatesynthetase <303> JOURNAL: J. Biochem. <304> VOLUME: 99 <305> ISSUE: 5<306> PAGES: 1455-1464 <307> DATE: 1986 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Wathelet, M., et al. <302> TITLE:Full-length sequence and expression of the 42 kDa 2-5A synthetase <303>JOURNAL: FEBS Lett. <304> VOLUME: 196 <305> ISSUE: 1 <306> PAGES:113-120 <307> DATE: 1986 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Benech, P., et al. <302> TITLE: Structure of two forms ofthe interferon-induced (2′-5′) oligo A synthetase of human cells <303>JOURNAL: EMBO J. <304> VOLUME: 4 <305> ISSUE: 9 <306> PAGES: 2249-2256<307> DATE: 1985 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Saunders, M.E., et al. <302> TITLE: Human 2-5A synthetase:characterization of a novel cDNA and corresponding gene structure <303>JOURNAL: EMBO J. <304> VOLUME: 4 <305> ISSUE: 7 <306> PAGES: 1761-1768<307> DATE: 1985 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Merlin, G., et al. <302> TITLE: Molecular cloning and sequenceof partial cDNA for interferon-induced (2′-5′)oligo(A_ synthetase mRNAfrom human cells <303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304>VOLUME: 80 <305> ISSUE: 16 <306> PAGES: 4904-4908 <307> DATE: 1983 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <400> SEQUENCE: 1 atg atg gat ctc aga aat acc cca gcc aaa tctctg gac aag ttc att 48 Met Met Asp Leu Arg Asn Thr Pro Ala Lys Ser LeuAsp Lys Phe Ile 1 5 10 15 gaa gac tat ctc ttg cca gac acg tgt ttc cgcatg caa atc gac cat 96 Glu Asp Tyr Leu Leu Pro Asp Thr Cys Phe Arg MetGln Ile Asp His 20 25 30 gcc att gac atc atc tgt ggg ttc ctg aag gaa aggtgc ttc cga ggt 144 Ala Ile Asp Ile Ile Cys Gly Phe Leu Lys Glu Arg CysPhe Arg Gly 35 40 45 agc tcc tac cct gtg tgt gtg tcc aag gtg gta aag ggtggc tcc tca 192 Ser Ser Tyr Pro Val Cys Val Ser Lys Val Val Lys Gly GlySer Ser 50 55 60 ggc aag ggc acc acc ctc aga ggc cga tct gac gct gac ctggtt gtc 240 Gly Lys Gly Thr Thr Leu Arg Gly Arg Ser Asp Ala Asp Leu ValVal 65 70 75 80 ttc ctc agt cct ctc acc act ttt cag gat cag tta aat cgccgg gga 288 Phe Leu Ser Pro Leu Thr Thr Phe Gln Asp Gln Leu Asn Arg ArgGly 85 90 95 gag ttc atc cag gaa att agg aga cag ctg gaa gcc tgt caa agagag 336 Glu Phe Ile Gln Glu Ile Arg Arg Gln Leu Glu Ala Cys Gln Arg Glu100 105 110 aga gca ctt tcc gtg aag ttt gag gtc cag gct cca cgc tgg ggcaac 384 Arg Ala Leu Ser Val Lys Phe Glu Val Gln Ala Pro Arg Trp Gly Asn115 120 125 ccc cgt gcg ctc agc ttc gta ctg agt tcg ctc cag ctc ggg gagggg 432 Pro Arg Ala Leu Ser Phe Val Leu Ser Ser Leu Gln Leu Gly Glu Gly130 135 140 gtg gag ttc gat gtg ctg cct gcc ttt gat gcc ctg ggt cag ttgact 480 Val Glu Phe Asp Val Leu Pro Ala Phe Asp Ala Leu Gly Gln Leu Thr145 150 155 160 ggc agc tat aaa cct aac ccc caa atc tat gtc aag ctc atcgag gag 528 Gly Ser Tyr Lys Pro Asn Pro Gln Ile Tyr Val Lys Leu Ile GluGlu 165 170 175 tgc acc gac ctg cag aaa gag ggc gag ttc tcc acc tgc ttcaca gaa 576 Cys Thr Asp Leu Gln Lys Glu Gly Glu Phe Ser Thr Cys Phe ThrGlu 180 185 190 cta cag aga gac ttc ctg aag cag cgc ccc acc aag ctc aagagc ctc 624 Leu Gln Arg Asp Phe Leu Lys Gln Arg Pro Thr Lys Leu Lys SerLeu 195 200 205 atc cgc cta gtc aag cac tgg tac caa aat tgt aag aag aagctt ggg 672 Ile Arg Leu Val Lys His Trp Tyr Gln Asn Cys Lys Lys Lys LeuGly 210 215 220 aag ctg cca cct cag tat gcc ctg gag ctc ctg acg gtc tatgct tgg 720 Lys Leu Pro Pro Gln Tyr Ala Leu Glu Leu Leu Thr Val Tyr AlaTrp 225 230 235 240 gag cga ggg agc atg aaa aca cat ttc aac aca gcc caagga ttt cgg 768 Glu Arg Gly Ser Met Lys Thr His Phe Asn Thr Ala Gln GlyPhe Arg 245 250 255 acg gtc ttg gaa tta gtc ata aac tac cag caa ctc tgcatc tac tgg 816 Thr Val Leu Glu Leu Val Ile Asn Tyr Gln Gln Leu Cys IleTyr Trp 260 265 270 aca aag tat tat gac ttt aaa aac ccc att att gaa aagtac ctg aga 864 Thr Lys Tyr Tyr Asp Phe Lys Asn Pro Ile Ile Glu Lys TyrLeu Arg 275 280 285 agg cag ctc acg aaa ccc agg cct gtg atc ctg gac ccggcg gac cct 912 Arg Gln Leu Thr Lys Pro Arg Pro Val Ile Leu Asp Pro AlaAsp Pro 290 295 300 aca gga aac ttg ggt ggt gga gac cca aag ggt tgg aggcag ctg gca 960 Thr Gly Asn Leu Gly Gly Gly Asp Pro Lys Gly Trp Arg GlnLeu Ala 305 310 315 320 caa gag gct gag gcc tgg ctg aat tac cca tgc tttaag aat tgg gat 1008 Gln Glu Ala Glu Ala Trp Leu Asn Tyr Pro Cys Phe LysAsn Trp Asp 325 330 335 ggg tcc cca gtg agc tcc tgg att ctg ctg gct gaaagc aac agt aca 1056 Gly Ser Pro Val Ser Ser Trp Ile Leu Leu Ala Glu SerAsn Ser Thr 340 345 350 gac gat gag acc gac gat ccc agg acg tat cag aaatat ggt tac att 1104 Asp Asp Glu Thr Asp Asp Pro Arg Thr Tyr Gln Lys TyrGly Tyr Ile 355 360 365 gga aca cat gag tac cct cat ttc tct cat aga cccagc acg ctc cag 1152 Gly Thr His Glu Tyr Pro His Phe Ser His Arg Pro SerThr Leu Gln 370 375 380 gca gca tcc acc cca cag gca gaa gag gac tgg acctgc acc atc ctc 1200 Ala Ala Ser Thr Pro Gln Ala Glu Glu Asp Trp Thr CysThr Ile Leu 385 390 395 400 tga 1203 <210> SEQ ID NO 2 <211> LENGTH: 400<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met MetAsp Leu Arg Asn Thr Pro Ala Lys Ser Leu Asp Lys Phe Ile 1 5 10 15 GluAsp Tyr Leu Leu Pro Asp Thr Cys Phe Arg Met Gln Ile Asp His 20 25 30 AlaIle Asp Ile Ile Cys Gly Phe Leu Lys Glu Arg Cys Phe Arg Gly 35 40 45 SerSer Tyr Pro Val Cys Val Ser Lys Val Val Lys Gly Gly Ser Ser 50 55 60 GlyLys Gly Thr Thr Leu Arg Gly Arg Ser Asp Ala Asp Leu Val Val 65 70 75 80Phe Leu Ser Pro Leu Thr Thr Phe Gln Asp Gln Leu Asn Arg Arg Gly 85 90 95Glu Phe Ile Gln Glu Ile Arg Arg Gln Leu Glu Ala Cys Gln Arg Glu 100 105110 Arg Ala Leu Ser Val Lys Phe Glu Val Gln Ala Pro Arg Trp Gly Asn 115120 125 Pro Arg Ala Leu Ser Phe Val Leu Ser Ser Leu Gln Leu Gly Glu Gly130 135 140 Val Glu Phe Asp Val Leu Pro Ala Phe Asp Ala Leu Gly Gln LeuThr 145 150 155 160 Gly Ser Tyr Lys Pro Asn Pro Gln Ile Tyr Val Lys LeuIle Glu Glu 165 170 175 Cys Thr Asp Leu Gln Lys Glu Gly Glu Phe Ser ThrCys Phe Thr Glu 180 185 190 Leu Gln Arg Asp Phe Leu Lys Gln Arg Pro ThrLys Leu Lys Ser Leu 195 200 205 Ile Arg Leu Val Lys His Trp Tyr Gln AsnCys Lys Lys Lys Leu Gly 210 215 220 Lys Leu Pro Pro Gln Tyr Ala Leu GluLeu Leu Thr Val Tyr Ala Trp 225 230 235 240 Glu Arg Gly Ser Met Lys ThrHis Phe Asn Thr Ala Gln Gly Phe Arg 245 250 255 Thr Val Leu Glu Leu ValIle Asn Tyr Gln Gln Leu Cys Ile Tyr Trp 260 265 270 Thr Lys Tyr Tyr AspPhe Lys Asn Pro Ile Ile Glu Lys Tyr Leu Arg 275 280 285 Arg Gln Leu ThrLys Pro Arg Pro Val Ile Leu Asp Pro Ala Asp Pro 290 295 300 Thr Gly AsnLeu Gly Gly Gly Asp Pro Lys Gly Trp Arg Gln Leu Ala 305 310 315 320 GlnGlu Ala Glu Ala Trp Leu Asn Tyr Pro Cys Phe Lys Asn Trp Asp 325 330 335Gly Ser Pro Val Ser Ser Trp Ile Leu Leu Ala Glu Ser Asn Ser Thr 340 345350 Asp Asp Glu Thr Asp Asp Pro Arg Thr Tyr Gln Lys Tyr Gly Tyr Ile 355360 365 Gly Thr His Glu Tyr Pro His Phe Ser His Arg Pro Ser Thr Leu Gln370 375 380 Ala Ala Ser Thr Pro Gln Ala Glu Glu Asp Trp Thr Cys Thr IleLeu 385 390 395 400 <210> SEQ ID NO 3 <211> LENGTH: 1207 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1206) <223> OTHER INFORMATION: <300> PUBLICATIONINFORMATION: <301> AUTHORS: Benech et al. <302> TITLE: Structure of twoforms of the interferon-induced (2′-5′) oligo A synthetase human cellsbased on cDNAs and gene sequences <303> JOURNAL: EMBO J. <304> VOLUME: 4<305> ISSUE: 9 <306> PAGES: 2249-2256 <307> DATE: 1985 <308> DATABASEACCESSION NUMBER: NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <400>SEQUENCE: 3 atg atg gat ctc aga aat acc cca gcc aaa tct ctg gac aag ttcatt 48 Met Met Asp Leu Arg Asn Thr Pro Ala Lys Ser Leu Asp Lys Phe Ile 15 10 15 gaa gac tat ctc ttg cca gac acg tgt ttc cgc atg caa atc gac cat96 Glu Asp Tyr Leu Leu Pro Asp Thr Cys Phe Arg Met Gln Ile Asp His 20 2530 gcc att gac atc atc tgt ggg ttc ctg aag gaa agg tgc ttc cga ggt 144Ala Ile Asp Ile Ile Cys Gly Phe Leu Lys Glu Arg Cys Phe Arg Gly 35 40 45agc tcc tac cct gtg tgt gtg tcc aag gtg gta aag ggt ggc tcc tca 192 SerSer Tyr Pro Val Cys Val Ser Lys Val Val Lys Gly Gly Ser Ser 50 55 60 ggcaag ggc acc acc ctc aga ggc cga tct gac gct gac ctg gtt gtc 240 Gly LysGly Thr Thr Leu Arg Gly Arg Ser Asp Ala Asp Leu Val Val 65 70 75 80 ttcctc agt cct ctc acc act ttt cag gat cag tta aat cgc cgg gga 288 Phe LeuSer Pro Leu Thr Thr Phe Gln Asp Gln Leu Asn Arg Arg Gly 85 90 95 gag ttcatc cag gaa att agg aga cag ctg gaa gcc tgt caa aga gag 336 Glu Phe IleGln Glu Ile Arg Arg Gln Leu Glu Ala Cys Gln Arg Glu 100 105 110 aga gcactt tcc gtg aag ttt gag gtc cag gct cca cgc tgg ggc aac 384 Arg Ala LeuSer Val Lys Phe Glu Val Gln Ala Pro Arg Trp Gly Asn 115 120 125 ccc cgtgcg ctc agc ttc gta ctg agt tcg ctc cag ctc ggg gag ggg 432 Pro Arg AlaLeu Ser Phe Val Leu Ser Ser Leu Gln Leu Gly Glu Gly 130 135 140 gtg gagttc gat gtg ctg cct gcc ttt gat gcc ctg ggt cag ttg act 480 Val Glu PheAsp Val Leu Pro Ala Phe Asp Ala Leu Gly Gln Leu Thr 145 150 155 160 ggcagc tat aaa cct aac ccc caa atc tat gtc aag ctc atc gag gag 528 Gly SerTyr Lys Pro Asn Pro Gln Ile Tyr Val Lys Leu Ile Glu Glu 165 170 175 tgcacc gac ctg cag aaa gag ggc gag ttc tcc acc tgc ttc aca gaa 576 Cys ThrAsp Leu Gln Lys Glu Gly Glu Phe Ser Thr Cys Phe Thr Glu 180 185 190 ctacag aga gac ttc ctg aag cag cgc ccc acc aag ctc aag agc ctc 624 Leu GlnArg Asp Phe Leu Lys Gln Arg Pro Thr Lys Leu Lys Ser Leu 195 200 205 atccgc cta gtc aag cac tgg tac caa aat tgt aag aag aag ctt ggg 672 Ile ArgLeu Val Lys His Trp Tyr Gln Asn Cys Lys Lys Lys Leu Gly 210 215 220 aagctg cca cct cag tat gcc ctg gag ctc ctg acg gtc tat gct tgg 720 Lys LeuPro Pro Gln Tyr Ala Leu Glu Leu Leu Thr Val Tyr Ala Trp 225 230 235 240gag cga ggg agc atg aaa aca cat ttc aac aca gcc caa gga ttt cgg 768 GluArg Gly Ser Met Lys Thr His Phe Asn Thr Ala Gln Gly Phe Arg 245 250 255acg gtc ttg gaa tta gtc ata aac tac cag caa ctc tgc atc tac tgg 816 ThrVal Leu Glu Leu Val Ile Asn Tyr Gln Gln Leu Cys Ile Tyr Trp 260 265 270aca aag tat tat gac ttt aaa aac ccc att att gaa aag tac ctg aga 864 ThrLys Tyr Tyr Asp Phe Lys Asn Pro Ile Ile Glu Lys Tyr Leu Arg 275 280 285agg cag ctc acg aaa ccc agg cct gtg atc ctg gac ccg gcg gac cct 912 ArgGln Leu Thr Lys Pro Arg Pro Val Ile Leu Asp Pro Ala Asp Pro 290 295 300aca gga aac ttg ggt ggt gga gac cca aag ggt tgg agg cag ctg gca 960 ThrGly Asn Leu Gly Gly Gly Asp Pro Lys Gly Trp Arg Gln Leu Ala 305 310 315320 caa gag gct gag gcc tgg ctg aat tac cca tgc ttt aag aat tgg gat 1008Gln Glu Ala Glu Ala Trp Leu Asn Tyr Pro Cys Phe Lys Asn Trp Asp 325 330335 ggg tcc cca gtg agc tcc tgg att ctg ctg gct gaa agc aac agt aca 1056Gly Ser Pro Val Ser Ser Trp Ile Leu Leu Ala Glu Ser Asn Ser Thr 340 345350 gac gat gag acc gac gat ccc agg acg tat cag aaa tat ggt tac att 1104Asp Asp Glu Thr Asp Asp Pro Arg Thr Tyr Gln Lys Tyr Gly Tyr Ile 355 360365 gga aca cat gag tac cct cat ttc tct cat aga ccc agc acg ctc cag 1152Gly Thr His Glu Tyr Pro His Phe Ser His Arg Pro Ser Thr Leu Gln 370 375380 gca gca tcc acc cca cag gca gaa gag gac tgg acc tgc acc atc ctc 1200Ala Ala Ser Thr Pro Gln Ala Glu Glu Asp Trp Thr Cys Thr Ile Leu 385 390395 400 tga atg c 1207 Met <210> SEQ ID NO 4 <211> LENGTH: 400 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Met Met Asp LeuArg Asn Thr Pro Ala Lys Ser Leu Asp Lys Phe Ile 1 5 10 15 Glu Asp TyrLeu Leu Pro Asp Thr Cys Phe Arg Met Gln Ile Asp His 20 25 30 Ala Ile AspIle Ile Cys Gly Phe Leu Lys Glu Arg Cys Phe Arg Gly 35 40 45 Ser Ser TyrPro Val Cys Val Ser Lys Val Val Lys Gly Gly Ser Ser 50 55 60 Gly Lys GlyThr Thr Leu Arg Gly Arg Ser Asp Ala Asp Leu Val Val 65 70 75 80 Phe LeuSer Pro Leu Thr Thr Phe Gln Asp Gln Leu Asn Arg Arg Gly 85 90 95 Glu PheIle Gln Glu Ile Arg Arg Gln Leu Glu Ala Cys Gln Arg Glu 100 105 110 ArgAla Leu Ser Val Lys Phe Glu Val Gln Ala Pro Arg Trp Gly Asn 115 120 125Pro Arg Ala Leu Ser Phe Val Leu Ser Ser Leu Gln Leu Gly Glu Gly 130 135140 Val Glu Phe Asp Val Leu Pro Ala Phe Asp Ala Leu Gly Gln Leu Thr 145150 155 160 Gly Ser Tyr Lys Pro Asn Pro Gln Ile Tyr Val Lys Leu Ile GluGlu 165 170 175 Cys Thr Asp Leu Gln Lys Glu Gly Glu Phe Ser Thr Cys PheThr Glu 180 185 190 Leu Gln Arg Asp Phe Leu Lys Gln Arg Pro Thr Lys LeuLys Ser Leu 195 200 205 Ile Arg Leu Val Lys His Trp Tyr Gln Asn Cys LysLys Lys Leu Gly 210 215 220 Lys Leu Pro Pro Gln Tyr Ala Leu Glu Leu LeuThr Val Tyr Ala Trp 225 230 235 240 Glu Arg Gly Ser Met Lys Thr His PheAsn Thr Ala Gln Gly Phe Arg 245 250 255 Thr Val Leu Glu Leu Val Ile AsnTyr Gln Gln Leu Cys Ile Tyr Trp 260 265 270 Thr Lys Tyr Tyr Asp Phe LysAsn Pro Ile Ile Glu Lys Tyr Leu Arg 275 280 285 Arg Gln Leu Thr Lys ProArg Pro Val Ile Leu Asp Pro Ala Asp Pro 290 295 300 Thr Gly Asn Leu GlyGly Gly Asp Pro Lys Gly Trp Arg Gln Leu Ala 305 310 315 320 Gln Glu AlaGlu Ala Trp Leu Asn Tyr Pro Cys Phe Lys Asn Trp Asp 325 330 335 Gly SerPro Val Ser Ser Trp Ile Leu Leu Ala Glu Ser Asn Ser Thr 340 345 350 AspAsp Glu Thr Asp Asp Pro Arg Thr Tyr Gln Lys Tyr Gly Tyr Ile 355 360 365Gly Thr His Glu Tyr Pro His Phe Ser His Arg Pro Ser Thr Leu Gln 370 375380 Ala Ala Ser Thr Pro Gln Ala Glu Glu Asp Trp Thr Cys Thr Ile Leu 385390 395 400 <210> SEQ ID NO 5 <211> LENGTH: 2064 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(2064) <223> OTHER INFORMATION: <300> PUBLICATIONINFORMATION: <301> AUTHORS: Hovnanian, A., et al. <302> TITLE: The human2′, 5′-oligoadenylate synthetase locus is comosed of three distinctgenes <303> JOURNAL: Genomics <304> VOLUME: 52 <305> ISSUE: 3 <306>PAGES: 267-277 <307> DATE: 1998 <308> DATABASE ACCESSION NUMBER:NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Marie, I. and Hovanessian, A.G. <302> TITLE:The 69-kDa 2-5A synthetase is composed of two homologous and adjacentfunctional domains <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 267 <305>ISSUE: 14 <306> PAGES: 9933-9939 <307> DATE: 1992 <308> DATABASEACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03<300> PUBLICATION INFORMATION: <301> AUTHORS: Marie, I., et al. <302>TITLE: Differential expression and distinct structure of 69- and 100-kDaforms of 2-5A synthetase <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 265<305> ISSUE: 30 <306> PAGES: 18601-18607 <307> DATE: 1990 <308> DATABASEACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03<300> PUBLICATION INFORMATION: <301> AUTHORS: Marie, I., et al. <302>TITLE: Preparation and characterization of polyclonal antibodiesspecific for the 69 and 100 k-dalton forms of human 2-5A synthetase<303> JOURNAL: Biochem. Biophys. Res. Commun. <304> VOLUME: 160 <305>ISSUE: 2 <306> PAGES: 580-587 <307> DATE: 1989 <308> DATABASE ACCESSIONNUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03 <300>PUBLICATION INFORMATION: <301> AUTHORS: Hovanessian, A.G., et al. <302>TITLE: Characterization of 69- and 100-kDa forms of 2-5A- synthetasefrom interferon-treated human cells <303> JOURNAL: J. Biol. Chem. <304>VOLUME: 263 <305> ISSUE: 10 <306> PAGES: 4959 <307> DATE: 1988 <308>DATABASE ACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE:2003-04-03 <300> PUBLICATION INFORMATION: <301> AUTHORS: Hovanessian,A.G., et al. <302> TITLE: Identification of 69-kd and 100-kd forms of2-5A synthesase <303> JOURNAL: EMBO J. <304> VOLUME: 6 <305> ISSUE: 5<306> PAGES: 1273-1280 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03 <400> SEQUENCE: 5atg gga aat ggg gag tcc cag ctg tcc tcg gtg cct gct cag aag ctg 48 MetGly Asn Gly Glu Ser Gln Leu Ser Ser Val Pro Ala Gln Lys Leu 1 5 10 15ggt tgg ttt atc cag gaa tac ctg aag ccc tac gaa gaa tgt cag aca 96 GlyTrp Phe Ile Gln Glu Tyr Leu Lys Pro Tyr Glu Glu Cys Gln Thr 20 25 30 ctgatc gac gag atg gtg aac acc atc tgt gac gtc tgc agg aac ccc 144 Leu IleAsp Glu Met Val Asn Thr Ile Cys Asp Val Cys Arg Asn Pro 35 40 45 gaa cagttc ccc ctg gtg cag gga gtg gcc ata ggt ggc tcc tat gga 192 Glu Gln PhePro Leu Val Gln Gly Val Ala Ile Gly Gly Ser Tyr Gly 50 55 60 cgg aaa acagtc tta aga ggc aac tcc gat ggt acc ctt gtc ctt ttc 240 Arg Lys Thr ValLeu Arg Gly Asn Ser Asp Gly Thr Leu Val Leu Phe 65 70 75 80 ttc agt gactta aaa caa ttc cag gat cag aag aga agc caa cgt gac 288 Phe Ser Asp LeuLys Gln Phe Gln Asp Gln Lys Arg Ser Gln Arg Asp 85 90 95 atc ctc gat aaaact ggg gat aag ctg aag ttc tgt ctg ttc acg aag 336 Ile Leu Asp Lys ThrGly Asp Lys Leu Lys Phe Cys Leu Phe Thr Lys 100 105 110 tgg ttg aaa aacaat ttc gag atc cag aag tcc ctt gat ggg tcc acc 384 Trp Leu Lys Asn AsnPhe Glu Ile Gln Lys Ser Leu Asp Gly Ser Thr 115 120 125 atc cag gtg ttcaca aaa aat cag aga atc tct ttc gag gtg ctg gcc 432 Ile Gln Val Phe ThrLys Asn Gln Arg Ile Ser Phe Glu Val Leu Ala 130 135 140 gcc ttc aac gctctg agc tta aat gat aat ccc agc ccc tgg atc tat 480 Ala Phe Asn Ala LeuSer Leu Asn Asp Asn Pro Ser Pro Trp Ile Tyr 145 150 155 160 cga gag ctcaaa aga tcc ttg gat aag aca aat gcc agt cct ggt gag 528 Arg Glu Leu LysArg Ser Leu Asp Lys Thr Asn Ala Ser Pro Gly Glu 165 170 175 ttt gca gtctgc ttc act gaa ctc cag cag aag ttt ttt gac aac cgt 576 Phe Ala Val CysPhe Thr Glu Leu Gln Gln Lys Phe Phe Asp Asn Arg 180 185 190 cct gga aaacta aag gat ttg atc ctc ttg ata aag cac tgg cat caa 624 Pro Gly Lys LeuLys Asp Leu Ile Leu Leu Ile Lys His Trp His Gln 195 200 205 cag tgc cagaaa aaa atc aag gat tta ccc tcg ctg tct ccg tat gcc 672 Gln Cys Gln LysLys Ile Lys Asp Leu Pro Ser Leu Ser Pro Tyr Ala 210 215 220 ctg gag ctgctt acg gtg tat gcc tgg gaa cag ggg tgc aga aaa gac 720 Leu Glu Leu LeuThr Val Tyr Ala Trp Glu Gln Gly Cys Arg Lys Asp 225 230 235 240 aac tttgac att gct gaa ggc gtc aga acg gtt ctg gag ctg atc aaa 768 Asn Phe AspIle Ala Glu Gly Val Arg Thr Val Leu Glu Leu Ile Lys 245 250 255 tgc caggag aag ctg tgt atc tat tgg atg gtc aac tac aac ttt gaa 816 Cys Gln GluLys Leu Cys Ile Tyr Trp Met Val Asn Tyr Asn Phe Glu 260 265 270 gat gagacc atc agg aac atc ctg ctg cac cag ctc caa tca gcg agg 864 Asp Glu ThrIle Arg Asn Ile Leu Leu His Gln Leu Gln Ser Ala Arg 275 280 285 cca gtaatc ttg gat cca gtt gac cca acc aat aat gtg agt gga gat 912 Pro Val IleLeu Asp Pro Val Asp Pro Thr Asn Asn Val Ser Gly Asp 290 295 300 aaa atatgc tgg caa tgg ctg aaa aaa gaa gct caa acc tgg ttg act 960 Lys Ile CysTrp Gln Trp Leu Lys Lys Glu Ala Gln Thr Trp Leu Thr 305 310 315 320 tctccc aac ctg gat aat gag tta cct gca cca tct tgg aat gtc ctg 1008 Ser ProAsn Leu Asp Asn Glu Leu Pro Ala Pro Ser Trp Asn Val Leu 325 330 335 cctgca cca ctc ttc acg acc cca ggc cac ctt ctg gat aag ttc atc 1056 Pro AlaPro Leu Phe Thr Thr Pro Gly His Leu Leu Asp Lys Phe Ile 340 345 350 aaggag ttt ctc cag ccc aac aaa tgc ttc cta gag cag att gac agt 1104 Lys GluPhe Leu Gln Pro Asn Lys Cys Phe Leu Glu Gln Ile Asp Ser 355 360 365 gctgtt aac atc atc cgt aca ttc ctt aaa gaa aac tgc ttc cga caa 1152 Ala ValAsn Ile Ile Arg Thr Phe Leu Lys Glu Asn Cys Phe Arg Gln 370 375 380 tcaaca gcc aag atc cag att gtc cgg gga gga tca acc gcc aaa ggc 1200 Ser ThrAla Lys Ile Gln Ile Val Arg Gly Gly Ser Thr Ala Lys Gly 385 390 395 400aca gct ctg aag act ggc tct gat gcc gat ctc gtc gtg ttc cat aac 1248 ThrAla Leu Lys Thr Gly Ser Asp Ala Asp Leu Val Val Phe His Asn 405 410 415tca ctt aaa agc tac acc tcc caa aaa aac gag cgg cac aaa atc gtc 1296 SerLeu Lys Ser Tyr Thr Ser Gln Lys Asn Glu Arg His Lys Ile Val 420 425 430aag gaa atc cat gaa cag ctg aaa gcc ttt tgg agg gag aag gag gag 1344 LysGlu Ile His Glu Gln Leu Lys Ala Phe Trp Arg Glu Lys Glu Glu 435 440 445gag ctt gaa gtc agc ttt gag cct ccc aag tgg aag gct ccc agg gtg 1392 GluLeu Glu Val Ser Phe Glu Pro Pro Lys Trp Lys Ala Pro Arg Val 450 455 460ctg agc ttc tct ctg aaa tcc aaa gtc ctc aac gaa agt gtc agc ttt 1440 LeuSer Phe Ser Leu Lys Ser Lys Val Leu Asn Glu Ser Val Ser Phe 465 470 475480 gat gtg ctt cct gcc ttt aat gca ctg ggt cag ctg agt tct ggc tcc 1488Asp Val Leu Pro Ala Phe Asn Ala Leu Gly Gln Leu Ser Ser Gly Ser 485 490495 aca ccc agc ccc gag gtt tat gca ggg ctc att gat ctg tat aaa tcc 1536Thr Pro Ser Pro Glu Val Tyr Ala Gly Leu Ile Asp Leu Tyr Lys Ser 500 505510 tcg gac ctc ccg gga gga gag ttt tct acc tgt ttc aca gtc ctg cag 1584Ser Asp Leu Pro Gly Gly Glu Phe Ser Thr Cys Phe Thr Val Leu Gln 515 520525 cga aac ttc att cgc tcc cgg ccc acc aaa cta aag gat tta att cgc 1632Arg Asn Phe Ile Arg Ser Arg Pro Thr Lys Leu Lys Asp Leu Ile Arg 530 535540 ctg gtg aag cac tgg tac aaa gag tgt gaa agg aaa ctg aag cca aag 1680Leu Val Lys His Trp Tyr Lys Glu Cys Glu Arg Lys Leu Lys Pro Lys 545 550555 560 ggg tct ttg ccc cca aag tat gcc ttg gag ctg ctc acc atc tat gcc1728 Gly Ser Leu Pro Pro Lys Tyr Ala Leu Glu Leu Leu Thr Ile Tyr Ala 565570 575 tgg gag cag ggg agt gga gtg ccg gat ttt gac act gca gaa ggt ttc1776 Trp Glu Gln Gly Ser Gly Val Pro Asp Phe Asp Thr Ala Glu Gly Phe 580585 590 cgg aca gtc ctg gag ctg gtc aca caa tat cag cag ctc ggc atc ttc1824 Arg Thr Val Leu Glu Leu Val Thr Gln Tyr Gln Gln Leu Gly Ile Phe 595600 605 tgg aag gtc aat tac aac ttt gaa gat gag acc gtg agg aag ttt cta1872 Trp Lys Val Asn Tyr Asn Phe Glu Asp Glu Thr Val Arg Lys Phe Leu 610615 620 ctg agc cag ttg cag aaa acc agg cct gtg atc ttg gac cca ggc gaa1920 Leu Ser Gln Leu Gln Lys Thr Arg Pro Val Ile Leu Asp Pro Gly Glu 625630 635 640 ccc aca ggt gac gtg ggt gga ggg gac cgt tgg tgt tgg cat cttctg 1968 Pro Thr Gly Asp Val Gly Gly Gly Asp Arg Trp Cys Trp His Leu Leu645 650 655 gac aaa gaa gca aag gtt agg tta tcc tct ccc tgc ttc aag gatggg 2016 Asp Lys Glu Ala Lys Val Arg Leu Ser Ser Pro Cys Phe Lys Asp Gly660 665 670 act gga aac cca ata cca cct tgg aaa gtg ccg gta aaa gtc atctaa 2064 Thr Gly Asn Pro Ile Pro Pro Trp Lys Val Pro Val Lys Val Ile 675680 685 <210> SEQ ID NO 6 <211> LENGTH: 687 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met Gly Asn Gly Glu Ser Gln LeuSer Ser Val Pro Ala Gln Lys Leu 1 5 10 15 Gly Trp Phe Ile Gln Glu TyrLeu Lys Pro Tyr Glu Glu Cys Gln Thr 20 25 30 Leu Ile Asp Glu Met Val AsnThr Ile Cys Asp Val Cys Arg Asn Pro 35 40 45 Glu Gln Phe Pro Leu Val GlnGly Val Ala Ile Gly Gly Ser Tyr Gly 50 55 60 Arg Lys Thr Val Leu Arg GlyAsn Ser Asp Gly Thr Leu Val Leu Phe 65 70 75 80 Phe Ser Asp Leu Lys GlnPhe Gln Asp Gln Lys Arg Ser Gln Arg Asp 85 90 95 Ile Leu Asp Lys Thr GlyAsp Lys Leu Lys Phe Cys Leu Phe Thr Lys 100 105 110 Trp Leu Lys Asn AsnPhe Glu Ile Gln Lys Ser Leu Asp Gly Ser Thr 115 120 125 Ile Gln Val PheThr Lys Asn Gln Arg Ile Ser Phe Glu Val Leu Ala 130 135 140 Ala Phe AsnAla Leu Ser Leu Asn Asp Asn Pro Ser Pro Trp Ile Tyr 145 150 155 160 ArgGlu Leu Lys Arg Ser Leu Asp Lys Thr Asn Ala Ser Pro Gly Glu 165 170 175Phe Ala Val Cys Phe Thr Glu Leu Gln Gln Lys Phe Phe Asp Asn Arg 180 185190 Pro Gly Lys Leu Lys Asp Leu Ile Leu Leu Ile Lys His Trp His Gln 195200 205 Gln Cys Gln Lys Lys Ile Lys Asp Leu Pro Ser Leu Ser Pro Tyr Ala210 215 220 Leu Glu Leu Leu Thr Val Tyr Ala Trp Glu Gln Gly Cys Arg LysAsp 225 230 235 240 Asn Phe Asp Ile Ala Glu Gly Val Arg Thr Val Leu GluLeu Ile Lys 245 250 255 Cys Gln Glu Lys Leu Cys Ile Tyr Trp Met Val AsnTyr Asn Phe Glu 260 265 270 Asp Glu Thr Ile Arg Asn Ile Leu Leu His GlnLeu Gln Ser Ala Arg 275 280 285 Pro Val Ile Leu Asp Pro Val Asp Pro ThrAsn Asn Val Ser Gly Asp 290 295 300 Lys Ile Cys Trp Gln Trp Leu Lys LysGlu Ala Gln Thr Trp Leu Thr 305 310 315 320 Ser Pro Asn Leu Asp Asn GluLeu Pro Ala Pro Ser Trp Asn Val Leu 325 330 335 Pro Ala Pro Leu Phe ThrThr Pro Gly His Leu Leu Asp Lys Phe Ile 340 345 350 Lys Glu Phe Leu GlnPro Asn Lys Cys Phe Leu Glu Gln Ile Asp Ser 355 360 365 Ala Val Asn IleIle Arg Thr Phe Leu Lys Glu Asn Cys Phe Arg Gln 370 375 380 Ser Thr AlaLys Ile Gln Ile Val Arg Gly Gly Ser Thr Ala Lys Gly 385 390 395 400 ThrAla Leu Lys Thr Gly Ser Asp Ala Asp Leu Val Val Phe His Asn 405 410 415Ser Leu Lys Ser Tyr Thr Ser Gln Lys Asn Glu Arg His Lys Ile Val 420 425430 Lys Glu Ile His Glu Gln Leu Lys Ala Phe Trp Arg Glu Lys Glu Glu 435440 445 Glu Leu Glu Val Ser Phe Glu Pro Pro Lys Trp Lys Ala Pro Arg Val450 455 460 Leu Ser Phe Ser Leu Lys Ser Lys Val Leu Asn Glu Ser Val SerPhe 465 470 475 480 Asp Val Leu Pro Ala Phe Asn Ala Leu Gly Gln Leu SerSer Gly Ser 485 490 495 Thr Pro Ser Pro Glu Val Tyr Ala Gly Leu Ile AspLeu Tyr Lys Ser 500 505 510 Ser Asp Leu Pro Gly Gly Glu Phe Ser Thr CysPhe Thr Val Leu Gln 515 520 525 Arg Asn Phe Ile Arg Ser Arg Pro Thr LysLeu Lys Asp Leu Ile Arg 530 535 540 Leu Val Lys His Trp Tyr Lys Glu CysGlu Arg Lys Leu Lys Pro Lys 545 550 555 560 Gly Ser Leu Pro Pro Lys TyrAla Leu Glu Leu Leu Thr Ile Tyr Ala 565 570 575 Trp Glu Gln Gly Ser GlyVal Pro Asp Phe Asp Thr Ala Glu Gly Phe 580 585 590 Arg Thr Val Leu GluLeu Val Thr Gln Tyr Gln Gln Leu Gly Ile Phe 595 600 605 Trp Lys Val AsnTyr Asn Phe Glu Asp Glu Thr Val Arg Lys Phe Leu 610 615 620 Leu Ser GlnLeu Gln Lys Thr Arg Pro Val Ile Leu Asp Pro Gly Glu 625 630 635 640 ProThr Gly Asp Val Gly Gly Gly Asp Arg Trp Cys Trp His Leu Leu 645 650 655Asp Lys Glu Ala Lys Val Arg Leu Ser Ser Pro Cys Phe Lys Asp Gly 660 665670 Thr Gly Asn Pro Ile Pro Pro Trp Lys Val Pro Val Lys Val Ile 675 680685 <210> SEQ ID NO 7 <211> LENGTH: 2186 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <300> PUBLICATION INFORMATION: <301> AUTHORS: Marie, I. andHovanessian, A.G. <302> TITLE: The 69-kDa 2-5A synthetase is composed oftwo homologous and adjacent functional domains <303> JOURNAL: J. Biol.Chem. <304> VOLUME: 267 <305> ISSUE: 14 <306> PAGES: 9933-9939 <307>DATE: 1992 <308> DATABASE ACCESSION NUMBER: (unknown) <309> DATABASEENTRY DATE: 2003-04-03 <400> SEQUENCE: 7 atgggaaatg gggagtcccagctgtcctcg gtgcctgctc agaagctggg ttggtttatc 60 caggaatacc tgaagccctacgaagaatgt cagacactga tcgacgagat ggtgaacacc 120 atctgtgacg tctgcaggaaccccgaacag ttccccctgg tgcagggagt ggccataggt 180 ggctcctatg gacggaaaacagtcttaaga ggcaactccg atggtaccct tgtccttttc 240 ttcagtgact taaaacaattccaggatcag aagagaagcc aacgtgacat cctcgataaa 300 actggggata agctgaagttctgtctgttc acgaagtggt tgaaaaacaa tttcgagatc 360 cagaagtccc ttgatgggtccaccatccag gtgttcacaa aaaatcagag aatctctttc 420 gaggtgctgg ccgccttcaacgctctgagc ttaaatgata atcccagccc ctggatctat 480 cgagagctca aaagatccttggataagaca aatgccagtc ctggtgagtt tgcagtctgc 540 ttcactgaac tccagcagaagttttttgac aaccgtcctg gaaaactaaa ggatttgatc 600 ctcttgataa agcactggcatcaacagtgc cagaaaaaaa tcaaggattt accctcgctg 660 tctccgtatg ccctggagctgcttacggtg tatgcctggg aacaggggtg cagaaaagac 720 aactttgaca ttgctgaaggcgtcagaacg gttctggagc tgatcaaatg ccaggagaag 780 ctgtgtatct attggatggtcaactacaac tttgaagatg agaccatcag gaacatcctg 840 ctgcaccagc tccaatcagcgaggccagta atcttggatc cagttgaccc aaccaataat 900 gtgagtggag ataaaatatgctggcaatgg ctgaaaaaag aagctcaaac ctggttgact 960 tctcccaacc tggataatgagttacctgca ccatcttgga atgtcctgcc tgcaccactc 1020 ttcacgaccc caggccaccttctggataag ttcatcaagg agtttctcca gcccaacaaa 1080 tgcttcctag agcagattgacagtgctgtt aacatcatcc gtacattcct taaagaaaac 1140 tgcttccgac aatcaacagccaagatccag attgtccggg gaggatcaac cgccaaaggc 1200 acagctctga agactggctctgatgccgat ctcgtcgtgt tccataactc acttaaaagc 1260 tacacctccc aaaaaaacgagcggcacaaa atcgtcaagg aaatccatga acagctgaaa 1320 gccttttgga gggagaaggaggaggagctt gaagtcagct ttgagcctcc caagtggaag 1380 gctcccaggg tgctgagcttctctctgaaa tccaaagtcc tcaacgaaag tgtcagcttt 1440 gatgtgcttc ctgcctttaatgcactgggt cagctgagtt ctggctccac acccagcccc 1500 gaggtttatg cagggctcattgatctgtat aaatcctcgg acctcccggg aggagagttt 1560 tctacctgtt tcacagtcctgcagcgaaac ttcattcgct cccggcccac caaactaaag 1620 gatttaattc gcctggtgaagcactggtac aaagagtgtg aaaggaaact gaagccaaag 1680 gggtctttgc ccccaaagtatgccttggag ctgctcacca tctatgcctg ggagcagggg 1740 agtggagtgc cggattttgacactgcagaa ggtttccgga cagtcctgga gctggtcaca 1800 caatatcagc agctcggcatcttctggaag gtcaattaca actttgaaga tgagaccgtg 1860 aggaagtttc tactgagccagttgcagaaa accaggcctg tgatcttgga cccaggcgaa 1920 cccacaggtg acgtgggtggaggggaccgt tggtgttggc atcttctgga caaagaagca 1980 aaggttaggt tatcctctccctgcttcaag gatgggactg gaaacccaat accaccttgg 2040 aaagtgccga caatgcagacaccaggaagt tgtggagcta ggattccatc ctattgtcaa 2100 tgagatgttg tcatccagaagccatagaat cctgaataat aattctaaaa gaaacttctg 2160 gagatcatct ggcaatcgcttttaaa 2186 <210> SEQ ID NO 8 <211> LENGTH: 727 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 8 Met Gly Asn Gly Glu Ser Gln LeuSer Ser Val Pro Ala Gln Lys Leu 1 5 10 15 Gly Trp Phe Ile Gln Glu TyrLeu Lys Pro Tyr Glu Glu Cys Gln Thr 20 25 30 Leu Ile Asp Glu Met Val AsnThr Ile Cys Asp Val Cys Arg Asn Pro 35 40 45 Glu Gln Phe Pro Leu Val GlnGly Val Ala Ile Gly Gly Ser Tyr Gly 50 55 60 Arg Lys Thr Val Leu Arg GlyAsn Ser Asp Gly Thr Leu Val Leu Phe 65 70 75 80 Phe Ser Asp Leu Lys GlnPhe Gln Asp Gln Lys Arg Ser Gln Arg Asp 85 90 95 Ile Leu Asp Lys Thr GlyAsp Lys Leu Lys Phe Cys Leu Phe Thr Lys 100 105 110 Trp Leu Lys Asn AsnPhe Glu Ile Gln Lys Ser Leu Asp Gly Ser Thr 115 120 125 Ile Gln Val PheThr Lys Asn Gln Arg Ile Ser Phe Glu Val Leu Ala 130 135 140 Ala Phe AsnAla Leu Ser Leu Asn Asp Asn Pro Ser Pro Trp Ile Tyr 145 150 155 160 ArgGlu Leu Lys Arg Ser Leu Asp Lys Thr Asn Ala Ser Pro Gly Glu 165 170 175Phe Ala Val Cys Phe Thr Glu Leu Gln Gln Lys Phe Phe Asp Asn Arg 180 185190 Pro Gly Lys Leu Lys Asp Leu Ile Leu Leu Ile Lys His Trp His Gln 195200 205 Gln Cys Gln Lys Lys Ile Lys Asp Leu Pro Ser Leu Ser Pro Tyr Ala210 215 220 Leu Glu Leu Leu Thr Val Tyr Ala Trp Glu Gln Gly Cys Arg LysAsp 225 230 235 240 Asn Phe Asp Ile Ala Glu Gly Val Arg Thr Val Leu GluLeu Ile Lys 245 250 255 Cys Gln Glu Lys Leu Cys Ile Tyr Trp Met Val AsnTyr Asn Phe Glu 260 265 270 Asp Glu Thr Ile Arg Asn Ile Leu Leu His GlnLeu Gln Ser Ala Arg 275 280 285 Pro Val Ile Leu Asp Pro Val Asp Pro ThrAsn Asn Val Ser Gly Asp 290 295 300 Lys Ile Cys Trp Gln Trp Leu Lys LysGlu Ala Gln Thr Trp Leu Thr 305 310 315 320 Ser Pro Asn Leu Asp Asn GluLeu Pro Ala Pro Ser Trp Asn Val Leu 325 330 335 Pro Ala Pro Leu Phe ThrThr Pro Gly His Leu Leu Asp Lys Phe Ile 340 345 350 Lys Glu Phe Leu GlnPro Asn Lys Cys Phe Leu Glu Gln Ile Asp Ser 355 360 365 Ala Val Asn IleIle Arg Thr Phe Leu Lys Glu Asn Cys Phe Arg Gln 370 375 380 Ser Thr AlaLys Ile Gln Ile Val Arg Gly Gly Ser Thr Ala Lys Gly 385 390 395 400 ThrAla Leu Lys Thr Gly Ser Asp Ala Asp Leu Val Val Phe His Asn 405 410 415Ser Leu Lys Ser Tyr Thr Ser Gln Lys Asn Glu Arg His Lys Ile Val 420 425430 Lys Glu Ile His Glu Gln Leu Lys Ala Phe Trp Arg Glu Lys Glu Glu 435440 445 Glu Leu Glu Val Ser Phe Glu Pro Pro Lys Trp Lys Ala Pro Arg Val450 455 460 Leu Ser Phe Ser Leu Lys Ser Lys Val Leu Asn Glu Ser Val SerPhe 465 470 475 480 Asp Val Leu Pro Ala Phe Asn Ala Leu Gly Gln Leu SerSer Gly Ser 485 490 495 Thr Pro Ser Pro Glu Val Tyr Ala Gly Leu Ile AspLeu Tyr Lys Ser 500 505 510 Ser Asp Leu Pro Gly Gly Glu Phe Ser Thr CysPhe Thr Val Leu Gln 515 520 525 Arg Asn Phe Ile Arg Ser Arg Pro Thr LysLeu Lys Asp Leu Ile Arg 530 535 540 Leu Val Lys His Trp Tyr Lys Glu CysGlu Arg Lys Leu Lys Pro Lys 545 550 555 560 Gly Ser Leu Pro Pro Lys TyrAla Leu Glu Leu Leu Thr Ile Tyr Ala 565 570 575 Trp Glu Gln Gly Ser GlyVal Pro Asp Phe Asp Thr Ala Glu Gly Phe 580 585 590 Arg Thr Val Leu GluLeu Val Thr Gln Tyr Gln Gln Leu Gly Ile Phe 595 600 605 Trp Lys Val AsnTyr Asn Phe Glu Asp Glu Thr Val Arg Lys Phe Leu 610 615 620 Leu Ser GlnLeu Gln Lys Thr Arg Pro Val Ile Leu Asp Pro Gly Glu 625 630 635 640 ProThr Gly Asp Val Gly Gly Gly Asp Arg Trp Cys Trp His Leu Leu 645 650 655Asp Lys Glu Ala Lys Val Arg Leu Ser Ser Pro Cys Phe Lys Asp Gly 660 665670 Thr Gly Asn Pro Ile Pro Pro Trp Lys Val Pro Thr Met Gln Thr Pro 675680 685 Gly Ser Cys Gly Ala Arg Ile His Pro Ile Val Asn Glu Met Phe Ser690 695 700 Ser Arg Ser His Arg Ile Leu Asn Asn Asn Ser Lys Arg Asn PheThr 705 710 715 720 Arg Ser Ser Gly Asn Arg Phe 725 <210> SEQ ID NO 9<211> LENGTH: 3264 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(3264) <223> OTHERINFORMATION: <300> PUBLICATION INFORMATION: <301> AUTHORS: Rebouillat,D., et al. <302> TITLE: The 100-kDa 2′,5′-oligoadenylate synthetasecatalyzing preferentially the synthesis of dimeric pppA2′p5′A molecules<303> JOURNAL: J. Biol. Chem. <304> VOLUME: 274 <305> ISSUE: 3 <306>PAGES: 1557-1565 <307> DATE: 1999 <308> DATABASE ACCESSION NUMBER:NCBI/AF_063613 <309> DATABASE ENTRY DATE: 1999-05-04 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Rebouillat, D. and Hovanessian, A.G. <302>TITLE: Direct Submission <303> JOURNAL: Submitted (07-May-1998) Dept.ofAids and Retroviruses, Institut Pasteur <304> VOLUME: 0 <305> ISSUE: 0<306> PAGES: 0 <307> DATE: 1998 <308> DATABASE ACCESSION NUMBER:NCBI/AF_063613 <309> DATABASE ENTRY DATE: 1999-05-04 <400> SEQUENCE: 9atg gac ttg tac agc acc ccg gcc gct gcg ctg gac agg ttc gtg gcc 48 MetAsp Leu Tyr Ser Thr Pro Ala Ala Ala Leu Asp Arg Phe Val Ala 1 5 10 15aga agg ctg cag ccg cgg aag gag ttc gta gag aag gcg cgg cgc gct 96 ArgArg Leu Gln Pro Arg Lys Glu Phe Val Glu Lys Ala Arg Arg Ala 20 25 30 ctgggc gcc ctg gcc gct gcc ctg agg gag cgc ggg ggc cgc ctc ggt 144 Leu GlyAla Leu Ala Ala Ala Leu Arg Glu Arg Gly Gly Arg Leu Gly 35 40 45 gct gctgcc ccg cgg gtg ctg aaa act gtc aag gga ggc tcc tcg ggc 192 Ala Ala AlaPro Arg Val Leu Lys Thr Val Lys Gly Gly Ser Ser Gly 50 55 60 cgg ggc acagct ctc aag ggt ggc tgt gat tct gaa ctt gtc atc ttc 240 Arg Gly Thr AlaLeu Lys Gly Gly Cys Asp Ser Glu Leu Val Ile Phe 65 70 75 80 ctc gac tgcttc aag agc tat gtg gac cag agg gcc cgc cgt gca gag 288 Leu Asp Cys PheLys Ser Tyr Val Asp Gln Arg Ala Arg Arg Ala Glu 85 90 95 atc ctc agt gagatg cgg gca tcg ctg gaa tcc tgg tgg cag aac cca 336 Ile Leu Ser Glu MetArg Ala Ser Leu Glu Ser Trp Trp Gln Asn Pro 100 105 110 gtc cct ggt ctgaga ctc acg ttt cct gag cag agc gtg cct ggg gcc 384 Val Pro Gly Leu ArgLeu Thr Phe Pro Glu Gln Ser Val Pro Gly Ala 115 120 125 ctg cag ttc cgcctg aca tcc gta gat ctt gag gac tgg atg gat gtt 432 Leu Gln Phe Arg LeuThr Ser Val Asp Leu Glu Asp Trp Met Asp Val 130 135 140 agc ctg gtg cctgcc ttc aat gtc ctg ggt cag gcc ggc tcc gcg gtc 480 Ser Leu Val Pro AlaPhe Asn Val Leu Gly Gln Ala Gly Ser Ala Val 145 150 155 160 aaa ccc aagcca caa gtc tac tct acc ctc ctc aac agt ggc tgc caa 528 Lys Pro Lys ProGln Val Tyr Ser Thr Leu Leu Asn Ser Gly Cys Gln 165 170 175 ggg ggc gagcat gcg gcc tgc ttc aca gag ctg cgg agg aac ttt gtg 576 Gly Gly Glu HisAla Ala Cys Phe Thr Glu Leu Arg Arg Asn Phe Val 180 185 190 aac att cgccca gcc aag ttg aag aac cta atc ttg ctg gtg aag cac 624 Asn Ile Arg ProAla Lys Leu Lys Asn Leu Ile Leu Leu Val Lys His 195 200 205 tgg tac caccag gtg tgc cta cag ggg ttg tgg aag gag acg ctg ccc 672 Trp Tyr His GlnVal Cys Leu Gln Gly Leu Trp Lys Glu Thr Leu Pro 210 215 220 ccg gtc tatgcc ctg gaa ttg ctg acc atc ttc gcc tgg gag cag ggc 720 Pro Val Tyr AlaLeu Glu Leu Leu Thr Ile Phe Ala Trp Glu Gln Gly 225 230 235 240 tgt aagaag gat gct ttc agc cta ggc gaa ggc ctc cga act gtc ctg 768 Cys Lys LysAsp Ala Phe Ser Leu Gly Glu Gly Leu Arg Thr Val Leu 245 250 255 ggc ctgatc caa cag cat cag cac ctg tgt gtt ttc tgg act gtc aac 816 Gly Leu IleGln Gln His Gln His Leu Cys Val Phe Trp Thr Val Asn 260 265 270 tat ggcttc gag gac cct gca gtt ggg cag ttc ttg cag cgg cac gtt 864 Tyr Gly PheGlu Asp Pro Ala Val Gly Gln Phe Leu Gln Arg His Val 275 280 285 aag agaccc agg cct gtg atc ctg gac cca gct gac ccc aca tgg gac 912 Lys Arg ProArg Pro Val Ile Leu Asp Pro Ala Asp Pro Thr Trp Asp 290 295 300 ctg gggaat ggg gca gcc tgg cac tgg gat ttg cat gcc cag gag gca 960 Leu Gly AsnGly Ala Ala Trp His Trp Asp Leu His Ala Gln Glu Ala 305 310 315 320 gcatcc tgc tat gac cac cca tgc ttt ctg agg ggg atg ggg gac cca 1008 Ala SerCys Tyr Asp His Pro Cys Phe Leu Arg Gly Met Gly Asp Pro 325 330 335 gtgcag tct tgg aag ggg ccg ggc ctt cca cgt gct gga tgc tca ggt 1056 Val GlnSer Trp Lys Gly Pro Gly Leu Pro Arg Ala Gly Cys Ser Gly 340 345 350 ttgggc cac ccc atc cag cta gac cct aac cag aag acc cct gaa aac 1104 Leu GlyHis Pro Ile Gln Leu Asp Pro Asn Gln Lys Thr Pro Glu Asn 355 360 365 agcaag agc ctc aat gct gtg tac cca aga gca ggg agc aaa cct ccc 1152 Ser LysSer Leu Asn Ala Val Tyr Pro Arg Ala Gly Ser Lys Pro Pro 370 375 380 tcatgc cca gct cct ggc ccc act gcg gag cca gca tcg tac ccc tct 1200 Ser CysPro Ala Pro Gly Pro Thr Ala Glu Pro Ala Ser Tyr Pro Ser 385 390 395 400gtg ccg gga atg gcc ttg gac ctg tct cag atc ccc acc aag gag ctg 1248 ValPro Gly Met Ala Leu Asp Leu Ser Gln Ile Pro Thr Lys Glu Leu 405 410 415gac cgc ttc atc cag gac cac ctg aag ccg agc ccc cag ttc cag gag 1296 AspArg Phe Ile Gln Asp His Leu Lys Pro Ser Pro Gln Phe Gln Glu 420 425 430cag gtg aaa aag gcc atc gac atc atc ttg cgc tgc ctc cat gag aac 1344 GlnVal Lys Lys Ala Ile Asp Ile Ile Leu Arg Cys Leu His Glu Asn 435 440 445tgt gtt cac aag gcc tca aga gtc agt aaa ggg ggc tca ttt ggc cgg 1392 CysVal His Lys Ala Ser Arg Val Ser Lys Gly Gly Ser Phe Gly Arg 450 455 460ggc aca gac cta agg gat ggc tgt gat gtt gaa ctc atc atc ttc ctc 1440 GlyThr Asp Leu Arg Asp Gly Cys Asp Val Glu Leu Ile Ile Phe Leu 465 470 475480 aac tgc ttc acg gac tac aag gac cag ggg ccc cgc cgc gca gag atc 1488Asn Cys Phe Thr Asp Tyr Lys Asp Gln Gly Pro Arg Arg Ala Glu Ile 485 490495 ctt gat gag atg cga gcg cac gta gaa tcc tgg tgg cag gac cag gtg 1536Leu Asp Glu Met Arg Ala His Val Glu Ser Trp Trp Gln Asp Gln Val 500 505510 ccc agc ctg agc ctt cag ttt cct gag cag aat gtg cct gag gct ctg 1584Pro Ser Leu Ser Leu Gln Phe Pro Glu Gln Asn Val Pro Glu Ala Leu 515 520525 cag ttc cag ctg gtg tcc aca gcc ctg aag agc tgg acg gat gtt agc 1632Gln Phe Gln Leu Val Ser Thr Ala Leu Lys Ser Trp Thr Asp Val Ser 530 535540 ctg ctg cct gcc ttc gat gct gtg ggg cag ctc agt tct ggc acc aaa 1680Leu Leu Pro Ala Phe Asp Ala Val Gly Gln Leu Ser Ser Gly Thr Lys 545 550555 560 cca aat ccc cag gtc tac tcg agg ctc ctc acc agt ggc tgc cag gag1728 Pro Asn Pro Gln Val Tyr Ser Arg Leu Leu Thr Ser Gly Cys Gln Glu 565570 575 ggc gag cat aag gcc tgc ttc gca gag ctg cgg agg aac ttc atg aac1776 Gly Glu His Lys Ala Cys Phe Ala Glu Leu Arg Arg Asn Phe Met Asn 580585 590 att cgc cct gtc aag ctg aag aac ctg att ctg ctg gtg aag cac tgg1824 Ile Arg Pro Val Lys Leu Lys Asn Leu Ile Leu Leu Val Lys His Trp 595600 605 tac cgc cag gtt gcg gct cag aac aaa gga aaa gga cca gcc cct gcc1872 Tyr Arg Gln Val Ala Ala Gln Asn Lys Gly Lys Gly Pro Ala Pro Ala 610615 620 tct ctg ccc cca gcc tat gcc ctg gag ctc ctc acc atc ttt gcc tgg1920 Ser Leu Pro Pro Ala Tyr Ala Leu Glu Leu Leu Thr Ile Phe Ala Trp 625630 635 640 gag cag ggc tgc agg cag gat tgt ttc aac atg gcc caa ggc ttccgg 1968 Glu Gln Gly Cys Arg Gln Asp Cys Phe Asn Met Ala Gln Gly Phe Arg645 650 655 acg gtg ctg ggg ctc gtg caa cag cat cag cag ctc tgt gtc tactgg 2016 Thr Val Leu Gly Leu Val Gln Gln His Gln Gln Leu Cys Val Tyr Trp660 665 670 acg gtc aac tat agc act gag gac cca gcc atg aga atg cac cttctt 2064 Thr Val Asn Tyr Ser Thr Glu Asp Pro Ala Met Arg Met His Leu Leu675 680 685 ggc cag ctt cga aaa ccc aga ccc ctg gtc ctg gac ccc gct gatccc 2112 Gly Gln Leu Arg Lys Pro Arg Pro Leu Val Leu Asp Pro Ala Asp Pro690 695 700 acc tgg aac gtg ggc cac ggt agc tgg gag ctg ttg gcc cag gaagca 2160 Thr Trp Asn Val Gly His Gly Ser Trp Glu Leu Leu Ala Gln Glu Ala705 710 715 720 gca gcg ctg ggg atg cag gcc tgc ttt ctg agt aga gac gggaca tct 2208 Ala Ala Leu Gly Met Gln Ala Cys Phe Leu Ser Arg Asp Gly ThrSer 725 730 735 gtg cag ccc tgg gat gtg atg cca gcc ctc ctt tac caa acccca gct 2256 Val Gln Pro Trp Asp Val Met Pro Ala Leu Leu Tyr Gln Thr ProAla 740 745 750 ggg gac ctt gac aag ttc atc agt gaa ttt ctc cag ccc aaccgc cag 2304 Gly Asp Leu Asp Lys Phe Ile Ser Glu Phe Leu Gln Pro Asn ArgGln 755 760 765 ttc ctg gcc cag gtg aac aag gcc gtt gat acc atc tgt tcattt ttg 2352 Phe Leu Ala Gln Val Asn Lys Ala Val Asp Thr Ile Cys Ser PheLeu 770 775 780 aag gaa aac tgc ttc cgg aat tct ccc atc aaa gtg atc aaggtg gtc 2400 Lys Glu Asn Cys Phe Arg Asn Ser Pro Ile Lys Val Ile Lys ValVal 785 790 795 800 aag ggt ggc tct tca gcc aaa ggc aca gct ctg cga ggccgc tca gat 2448 Lys Gly Gly Ser Ser Ala Lys Gly Thr Ala Leu Arg Gly ArgSer Asp 805 810 815 gcc gac ctc gtg gtg ttc ctc agc tgc ttc agc cag ttcact gag cag 2496 Ala Asp Leu Val Val Phe Leu Ser Cys Phe Ser Gln Phe ThrGlu Gln 820 825 830 ggc aac aag cgg gcc gag atc atc tcc gag atc cga gcccag ctg gag 2544 Gly Asn Lys Arg Ala Glu Ile Ile Ser Glu Ile Arg Ala GlnLeu Glu 835 840 845 gca tgt caa cag gag cgg cag ttc gag gtc aag ttt gaagtc tcc aaa 2592 Ala Cys Gln Gln Glu Arg Gln Phe Glu Val Lys Phe Glu ValSer Lys 850 855 860 tgg gag aat ccc cgc gtg ctg agc ttc tca ctg aca tcccag acg atg 2640 Trp Glu Asn Pro Arg Val Leu Ser Phe Ser Leu Thr Ser GlnThr Met 865 870 875 880 ctg gac cag agt gtg gac ttt gat gtg ctg cca gccttt gac gcc cta 2688 Leu Asp Gln Ser Val Asp Phe Asp Val Leu Pro Ala PheAsp Ala Leu 885 890 895 ggc cag ctg gtc tct ggc tcc agg ccc agc tct caagtc tac gtc gac 2736 Gly Gln Leu Val Ser Gly Ser Arg Pro Ser Ser Gln ValTyr Val Asp 900 905 910 ctc atc cac agc tac agc aat gcg ggc gag tac tccacc tgc ttc aca 2784 Leu Ile His Ser Tyr Ser Asn Ala Gly Glu Tyr Ser ThrCys Phe Thr 915 920 925 gag cta caa cgg gac ttc atc atc tct cgc cct accaag ctg aag agc 2832 Glu Leu Gln Arg Asp Phe Ile Ile Ser Arg Pro Thr LysLeu Lys Ser 930 935 940 ctg atc cgg ctg gtg aag cac tgg tac cag cag tgtacc aag atc tcc 2880 Leu Ile Arg Leu Val Lys His Trp Tyr Gln Gln Cys ThrLys Ile Ser 945 950 955 960 aag ggg aga ggc tcc cta ccc cca cag cac gggctg gaa ctc ctg act 2928 Lys Gly Arg Gly Ser Leu Pro Pro Gln His Gly LeuGlu Leu Leu Thr 965 970 975 gtg tat gcc tgg gag cag ggc ggg aag gac tcccag ttc aac atg gct 2976 Val Tyr Ala Trp Glu Gln Gly Gly Lys Asp Ser GlnPhe Asn Met Ala 980 985 990 gag ggc ttc cgc acg gtc ctg gag ctg gtc acccag tac cgc cag ctc 3024 Glu Gly Phe Arg Thr Val Leu Glu Leu Val Thr GlnTyr Arg Gln Leu 995 1000 1005 tgt atc tac tgg acc atc aac tac aac gccaag gac aag act gtt 3069 Cys Ile Tyr Trp Thr Ile Asn Tyr Asn Ala Lys AspLys Thr Val 1010 1015 1020 gga gac ttc ctg aaa cag cag ctt cag aag cccagg cct atc atc 3114 Gly Asp Phe Leu Lys Gln Gln Leu Gln Lys Pro Arg ProIle Ile 1025 1030 1035 ctg gat ccg gct gac ccg aca ggc aac ctg ggc cacaat gcc cgc 3159 Leu Asp Pro Ala Asp Pro Thr Gly Asn Leu Gly His Asn AlaArg 1040 1045 1050 tgg gac ctg ctg gcc aag gaa gct gca gcc tgc aca tctgcc ctg 3204 Trp Asp Leu Leu Ala Lys Glu Ala Ala Ala Cys Thr Ser Ala Leu1055 1060 1065 tgc tgc atg gga cgg aat ggc atc ccc atc cag cca tgg ccagtg 3249 Cys Cys Met Gly Arg Asn Gly Ile Pro Ile Gln Pro Trp Pro Val1070 1075 1080 aag gct gct gtg tga 3264 Lys Ala Ala Val 1085 <210> SEQID NO 10 <211> LENGTH: 1087 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 10 Met Asp Leu Tyr Ser Thr Pro Ala Ala Ala Leu Asp ArgPhe Val Ala 1 5 10 15 Arg Arg Leu Gln Pro Arg Lys Glu Phe Val Glu LysAla Arg Arg Ala 20 25 30 Leu Gly Ala Leu Ala Ala Ala Leu Arg Glu Arg GlyGly Arg Leu Gly 35 40 45 Ala Ala Ala Pro Arg Val Leu Lys Thr Val Lys GlyGly Ser Ser Gly 50 55 60 Arg Gly Thr Ala Leu Lys Gly Gly Cys Asp Ser GluLeu Val Ile Phe 65 70 75 80 Leu Asp Cys Phe Lys Ser Tyr Val Asp Gln ArgAla Arg Arg Ala Glu 85 90 95 Ile Leu Ser Glu Met Arg Ala Ser Leu Glu SerTrp Trp Gln Asn Pro 100 105 110 Val Pro Gly Leu Arg Leu Thr Phe Pro GluGln Ser Val Pro Gly Ala 115 120 125 Leu Gln Phe Arg Leu Thr Ser Val AspLeu Glu Asp Trp Met Asp Val 130 135 140 Ser Leu Val Pro Ala Phe Asn ValLeu Gly Gln Ala Gly Ser Ala Val 145 150 155 160 Lys Pro Lys Pro Gln ValTyr Ser Thr Leu Leu Asn Ser Gly Cys Gln 165 170 175 Gly Gly Glu His AlaAla Cys Phe Thr Glu Leu Arg Arg Asn Phe Val 180 185 190 Asn Ile Arg ProAla Lys Leu Lys Asn Leu Ile Leu Leu Val Lys His 195 200 205 Trp Tyr HisGln Val Cys Leu Gln Gly Leu Trp Lys Glu Thr Leu Pro 210 215 220 Pro ValTyr Ala Leu Glu Leu Leu Thr Ile Phe Ala Trp Glu Gln Gly 225 230 235 240Cys Lys Lys Asp Ala Phe Ser Leu Gly Glu Gly Leu Arg Thr Val Leu 245 250255 Gly Leu Ile Gln Gln His Gln His Leu Cys Val Phe Trp Thr Val Asn 260265 270 Tyr Gly Phe Glu Asp Pro Ala Val Gly Gln Phe Leu Gln Arg His Val275 280 285 Lys Arg Pro Arg Pro Val Ile Leu Asp Pro Ala Asp Pro Thr TrpAsp 290 295 300 Leu Gly Asn Gly Ala Ala Trp His Trp Asp Leu His Ala GlnGlu Ala 305 310 315 320 Ala Ser Cys Tyr Asp His Pro Cys Phe Leu Arg GlyMet Gly Asp Pro 325 330 335 Val Gln Ser Trp Lys Gly Pro Gly Leu Pro ArgAla Gly Cys Ser Gly 340 345 350 Leu Gly His Pro Ile Gln Leu Asp Pro AsnGln Lys Thr Pro Glu Asn 355 360 365 Ser Lys Ser Leu Asn Ala Val Tyr ProArg Ala Gly Ser Lys Pro Pro 370 375 380 Ser Cys Pro Ala Pro Gly Pro ThrAla Glu Pro Ala Ser Tyr Pro Ser 385 390 395 400 Val Pro Gly Met Ala LeuAsp Leu Ser Gln Ile Pro Thr Lys Glu Leu 405 410 415 Asp Arg Phe Ile GlnAsp His Leu Lys Pro Ser Pro Gln Phe Gln Glu 420 425 430 Gln Val Lys LysAla Ile Asp Ile Ile Leu Arg Cys Leu His Glu Asn 435 440 445 Cys Val HisLys Ala Ser Arg Val Ser Lys Gly Gly Ser Phe Gly Arg 450 455 460 Gly ThrAsp Leu Arg Asp Gly Cys Asp Val Glu Leu Ile Ile Phe Leu 465 470 475 480Asn Cys Phe Thr Asp Tyr Lys Asp Gln Gly Pro Arg Arg Ala Glu Ile 485 490495 Leu Asp Glu Met Arg Ala His Val Glu Ser Trp Trp Gln Asp Gln Val 500505 510 Pro Ser Leu Ser Leu Gln Phe Pro Glu Gln Asn Val Pro Glu Ala Leu515 520 525 Gln Phe Gln Leu Val Ser Thr Ala Leu Lys Ser Trp Thr Asp ValSer 530 535 540 Leu Leu Pro Ala Phe Asp Ala Val Gly Gln Leu Ser Ser GlyThr Lys 545 550 555 560 Pro Asn Pro Gln Val Tyr Ser Arg Leu Leu Thr SerGly Cys Gln Glu 565 570 575 Gly Glu His Lys Ala Cys Phe Ala Glu Leu ArgArg Asn Phe Met Asn 580 585 590 Ile Arg Pro Val Lys Leu Lys Asn Leu IleLeu Leu Val Lys His Trp 595 600 605 Tyr Arg Gln Val Ala Ala Gln Asn LysGly Lys Gly Pro Ala Pro Ala 610 615 620 Ser Leu Pro Pro Ala Tyr Ala LeuGlu Leu Leu Thr Ile Phe Ala Trp 625 630 635 640 Glu Gln Gly Cys Arg GlnAsp Cys Phe Asn Met Ala Gln Gly Phe Arg 645 650 655 Thr Val Leu Gly LeuVal Gln Gln His Gln Gln Leu Cys Val Tyr Trp 660 665 670 Thr Val Asn TyrSer Thr Glu Asp Pro Ala Met Arg Met His Leu Leu 675 680 685 Gly Gln LeuArg Lys Pro Arg Pro Leu Val Leu Asp Pro Ala Asp Pro 690 695 700 Thr TrpAsn Val Gly His Gly Ser Trp Glu Leu Leu Ala Gln Glu Ala 705 710 715 720Ala Ala Leu Gly Met Gln Ala Cys Phe Leu Ser Arg Asp Gly Thr Ser 725 730735 Val Gln Pro Trp Asp Val Met Pro Ala Leu Leu Tyr Gln Thr Pro Ala 740745 750 Gly Asp Leu Asp Lys Phe Ile Ser Glu Phe Leu Gln Pro Asn Arg Gln755 760 765 Phe Leu Ala Gln Val Asn Lys Ala Val Asp Thr Ile Cys Ser PheLeu 770 775 780 Lys Glu Asn Cys Phe Arg Asn Ser Pro Ile Lys Val Ile LysVal Val 785 790 795 800 Lys Gly Gly Ser Ser Ala Lys Gly Thr Ala Leu ArgGly Arg Ser Asp 805 810 815 Ala Asp Leu Val Val Phe Leu Ser Cys Phe SerGln Phe Thr Glu Gln 820 825 830 Gly Asn Lys Arg Ala Glu Ile Ile Ser GluIle Arg Ala Gln Leu Glu 835 840 845 Ala Cys Gln Gln Glu Arg Gln Phe GluVal Lys Phe Glu Val Ser Lys 850 855 860 Trp Glu Asn Pro Arg Val Leu SerPhe Ser Leu Thr Ser Gln Thr Met 865 870 875 880 Leu Asp Gln Ser Val AspPhe Asp Val Leu Pro Ala Phe Asp Ala Leu 885 890 895 Gly Gln Leu Val SerGly Ser Arg Pro Ser Ser Gln Val Tyr Val Asp 900 905 910 Leu Ile His SerTyr Ser Asn Ala Gly Glu Tyr Ser Thr Cys Phe Thr 915 920 925 Glu Leu GlnArg Asp Phe Ile Ile Ser Arg Pro Thr Lys Leu Lys Ser 930 935 940 Leu IleArg Leu Val Lys His Trp Tyr Gln Gln Cys Thr Lys Ile Ser 945 950 955 960Lys Gly Arg Gly Ser Leu Pro Pro Gln His Gly Leu Glu Leu Leu Thr 965 970975 Val Tyr Ala Trp Glu Gln Gly Gly Lys Asp Ser Gln Phe Asn Met Ala 980985 990 Glu Gly Phe Arg Thr Val Leu Glu Leu Val Thr Gln Tyr Arg Gln Leu995 1000 1005 Cys Ile Tyr Trp Thr Ile Asn Tyr Asn Ala Lys Asp Lys ThrVal 1010 1015 1020 Gly Asp Phe Leu Lys Gln Gln Leu Gln Lys Pro Arg ProIle Ile 1025 1030 1035 Leu Asp Pro Ala Asp Pro Thr Gly Asn Leu Gly HisAsn Ala Arg 1040 1045 1050 Trp Asp Leu Leu Ala Lys Glu Ala Ala Ala CysThr Ser Ala Leu 1055 1060 1065 Cys Cys Met Gly Arg Asn Gly Ile Pro IleGln Pro Trp Pro Val 1070 1075 1080 Lys Ala Ala Val 1085 <210> SEQ ID NO11 <211> LENGTH: 1104 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1104) <223> OTHERINFORMATION: <300> PUBLICATION INFORMATION: <301> AUTHORS: Coccia, E.M.,et al. <302> TITLE: A full-length murine 2-5A synthetase cDNAtransfected in NIH-3T3 cells <303> JOURNAL: Virology <304> VOLUME: 179<305> ISSUE: 1 <306> PAGES: 228-233 <307> DATE: 1990 <308> DATABASEACCESSION NUMBER: NCBI/M33863 <309> DATABASE ENTRY DATE: 1993-06-11<400> SEQUENCE: 11 atg gag cac gga ctc agg agc atc cca gcc tgg acg ctggac aag ttc 48 Met Glu His Gly Leu Arg Ser Ile Pro Ala Trp Thr Leu AspLys Phe 1 5 10 15 ata gag gat tac ctc ctt ccc gac acc acc ttt ggt gctgat gtc aaa 96 Ile Glu Asp Tyr Leu Leu Pro Asp Thr Thr Phe Gly Ala AspVal Lys 20 25 30 tca gcc gtc aat gtc gtg tgt gat ttc ctg aag gag aga tgcttc caa 144 Ser Ala Val Asn Val Val Cys Asp Phe Leu Lys Glu Arg Cys PheGln 35 40 45 ggt gct gcc cac cca gtg agg gtc tcc aag gtg gtg aag ggt ggctcc 192 Gly Ala Ala His Pro Val Arg Val Ser Lys Val Val Lys Gly Gly Ser50 55 60 tca ggc aaa ggc acc aca ctc aag ggc agg tca gac gct gac ctg gtg240 Ser Gly Lys Gly Thr Thr Leu Lys Gly Arg Ser Asp Ala Asp Leu Val 6570 75 80 gtg ttc ctt aac aat ctc acc agc ttt gag gat cag tta aac cga cgg288 Val Phe Leu Asn Asn Leu Thr Ser Phe Glu Asp Gln Leu Asn Arg Arg 8590 95 gga gag ttc atc aag gaa att aag aaa cag ctg tac gag gtt cag cat336 Gly Glu Phe Ile Lys Glu Ile Lys Lys Gln Leu Tyr Glu Val Gln His 100105 110 gag aga cgt ttt aga gtc aag ttt gag gtc cag agt tca tgg tgg ccc384 Glu Arg Arg Phe Arg Val Lys Phe Glu Val Gln Ser Ser Trp Trp Pro 115120 125 aac gcc cgg tct ctg agc ttc aag ctg agc gcc ccc cat ctg cat cag432 Asn Ala Arg Ser Leu Ser Phe Lys Leu Ser Ala Pro His Leu His Gln 130135 140 gag gtg gag ttt gat gtg ctg cca gcc ttt gat gtc ctg ggt cat gtt480 Glu Val Glu Phe Asp Val Leu Pro Ala Phe Asp Val Leu Gly His Val 145150 155 160 aat act tcc agc aag cct gat ccc aga atc tat gcc atc ctc atcgag 528 Asn Thr Ser Ser Lys Pro Asp Pro Arg Ile Tyr Ala Ile Leu Ile Glu165 170 175 gaa tgt acc tcc ctg ggg aag gat ggc gag ttc tct acc tgc ttcacg 576 Glu Cys Thr Ser Leu Gly Lys Asp Gly Glu Phe Ser Thr Cys Phe Thr180 185 190 gag ctc cag cgg aac ttc ctg aag cag cgc cca acc aag ctg aagagt 624 Glu Leu Gln Arg Asn Phe Leu Lys Gln Arg Pro Thr Lys Leu Lys Ser195 200 205 ctc atc cgc ctg gtc aag cac tgg tac caa ctg tgt aag gag aagctg 672 Leu Ile Arg Leu Val Lys His Trp Tyr Gln Leu Cys Lys Glu Lys Leu210 215 220 ggg aag cca ttg cct cca cag tac gcc cta gag ttg ctc act gtcttt 720 Gly Lys Pro Leu Pro Pro Gln Tyr Ala Leu Glu Leu Leu Thr Val Phe225 230 235 240 gcc tgg gaa caa ggg aat gga tgt tat gag ttc aac aca gcccag ggc 768 Ala Trp Glu Gln Gly Asn Gly Cys Tyr Glu Phe Asn Thr Ala GlnGly 245 250 255 ttc cgg acc gtc ttg gaa ctg gtc atc aat tat cag cat cttcga atc 816 Phe Arg Thr Val Leu Glu Leu Val Ile Asn Tyr Gln His Leu ArgIle 260 265 270 tac tgg aca aag tat tat gac ttt caa cac cag gag gtc tccaaa tac 864 Tyr Trp Thr Lys Tyr Tyr Asp Phe Gln His Gln Glu Val Ser LysTyr 275 280 285 ctg cac aga cag ctc aga aaa gcc agg cct gtg atc ctg gaccca gct 912 Leu His Arg Gln Leu Arg Lys Ala Arg Pro Val Ile Leu Asp ProAla 290 295 300 gac cca aca ggg aat gtg gcc ggt ggg aac cca gag ggc tggagg cgg 960 Asp Pro Thr Gly Asn Val Ala Gly Gly Asn Pro Glu Gly Trp ArgArg 305 310 315 320 ttg gct gaa gag gct gat gtg tgg cta tgg tac cca tgtttt att aaa 1008 Leu Ala Glu Glu Ala Asp Val Trp Leu Trp Tyr Pro Cys PheIle Lys 325 330 335 aag gat ggt tcc cga gtg agc tcc tgg gat gtg ccg acggtg gtt cct 1056 Lys Asp Gly Ser Arg Val Ser Ser Trp Asp Val Pro Thr ValVal Pro 340 345 350 gta cct ttt gag cag gta gaa gag aac tgg aca tgt atcctg ctg tga 1104 Val Pro Phe Glu Gln Val Glu Glu Asn Trp Thr Cys Ile LeuLeu 355 360 365 <210> SEQ ID NO 12 <211> LENGTH: 367 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 12 Met Glu His Gly Leu ArgSer Ile Pro Ala Trp Thr Leu Asp Lys Phe 1 5 10 15 Ile Glu Asp Tyr LeuLeu Pro Asp Thr Thr Phe Gly Ala Asp Val Lys 20 25 30 Ser Ala Val Asn ValVal Cys Asp Phe Leu Lys Glu Arg Cys Phe Gln 35 40 45 Gly Ala Ala His ProVal Arg Val Ser Lys Val Val Lys Gly Gly Ser 50 55 60 Ser Gly Lys Gly ThrThr Leu Lys Gly Arg Ser Asp Ala Asp Leu Val 65 70 75 80 Val Phe Leu AsnAsn Leu Thr Ser Phe Glu Asp Gln Leu Asn Arg Arg 85 90 95 Gly Glu Phe IleLys Glu Ile Lys Lys Gln Leu Tyr Glu Val Gln His 100 105 110 Glu Arg ArgPhe Arg Val Lys Phe Glu Val Gln Ser Ser Trp Trp Pro 115 120 125 Asn AlaArg Ser Leu Ser Phe Lys Leu Ser Ala Pro His Leu His Gln 130 135 140 GluVal Glu Phe Asp Val Leu Pro Ala Phe Asp Val Leu Gly His Val 145 150 155160 Asn Thr Ser Ser Lys Pro Asp Pro Arg Ile Tyr Ala Ile Leu Ile Glu 165170 175 Glu Cys Thr Ser Leu Gly Lys Asp Gly Glu Phe Ser Thr Cys Phe Thr180 185 190 Glu Leu Gln Arg Asn Phe Leu Lys Gln Arg Pro Thr Lys Leu LysSer 195 200 205 Leu Ile Arg Leu Val Lys His Trp Tyr Gln Leu Cys Lys GluLys Leu 210 215 220 Gly Lys Pro Leu Pro Pro Gln Tyr Ala Leu Glu Leu LeuThr Val Phe 225 230 235 240 Ala Trp Glu Gln Gly Asn Gly Cys Tyr Glu PheAsn Thr Ala Gln Gly 245 250 255 Phe Arg Thr Val Leu Glu Leu Val Ile AsnTyr Gln His Leu Arg Ile 260 265 270 Tyr Trp Thr Lys Tyr Tyr Asp Phe GlnHis Gln Glu Val Ser Lys Tyr 275 280 285 Leu His Arg Gln Leu Arg Lys AlaArg Pro Val Ile Leu Asp Pro Ala 290 295 300 Asp Pro Thr Gly Asn Val AlaGly Gly Asn Pro Glu Gly Trp Arg Arg 305 310 315 320 Leu Ala Glu Glu AlaAsp Val Trp Leu Trp Tyr Pro Cys Phe Ile Lys 325 330 335 Lys Asp Gly SerArg Val Ser Ser Trp Asp Val Pro Thr Val Val Pro 340 345 350 Val Pro PheGlu Gln Val Glu Glu Asn Trp Thr Cys Ile Leu Leu 355 360 365 <210> SEQ IDNO 13 <211> LENGTH: 1590 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<300> PUBLICATION INFORMATION: <301> AUTHORS: Aissouni, Y. et al. <302>TITLE: The cleavage/polyadenylation activity triggered by a U-rich motifsequence <303> JOURNAL: J. Biol. Chem. <304> VOLUME: 277 <305> ISSUE: 39<306> PAGES: 35808-35814 <307> DATE: 2002 <308> DATABASE ACCESSIONNUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300>PUBLICATION INFORMATION: <301> AUTHORS: Behera, A.K., et al. <302>TITLE: 2′-5′ Oligoadenylate synthetase plays a critical role ininterferon-gamma inhibition <303> JOURNAL: J. Biol. Chem. <304> VOLUME:277 <305> ISSUE: 28 <306> PAGES: 25601-25608 <307> DATE: 2002 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Sarkar, S.N.,et al. <302> TITLE: Identification of the substrate-binding sites of2′-5′- oligoadenylate synthetase <303> JOURNAL: J. Biol. Chem. <304>VOLUME: 277 <305> ISSUE: 27 <306> PAGES: 24321-24330 <307> DATE: 2002<308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRYDATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS:Hovnanian, A., et al. <302> TITLE: The human 2′,5′-oligoadenylatesynthetase locus is composed of three distinct genes <303> JOURNAL:Genomics <304> VOLUME: 52 <305> ISSUE: 3 <306> PAGES: 267-277 <307>DATE: 1998 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Renault, B. <302> TITLE: A sequence-ready physical map of aregion of 12q24.1 <303> JOURNAL: Genomics <304> VOLUME: 45 <305> ISSUE:2 <306> PAGES: 271-278 <307> DATE: 1997 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Nechiporuk, T., et al. <302> TITLE: Ahigh-resolution PAC and BAC map of the SCA2 region <303> JOURNAL:Genomics <304> VOLUME: 44 <305> ISSUE: 3 <306> PAGES: 321-329 <307>DATE: 1997 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309>DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301>AUTHORS: Wathelet, M.G., et al. <302> TITLE: Cloning and chromosomallocation of human genes inducible by type I interferon <303> JOURNAL:Somat. Cell Mol. Genet. <304> VOLUME: 14 <305> ISSUE: 5 <306> PAGES:415-426 <307> DATE: 1988 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Rutherford, M.N., et al. <302> TITLE: Interferon-inducedbinding of nuclear factors to promoter elements of the 2-5A synthetasegene <303> JOURNAL: EMBO J. <304> VOLUME: 7 <305> ISSUE: 3 <306> PAGES:751-759 <307> DATE: 1988 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Wathelet, M.G., et al. <302> TITLE: New inducers revealedby the promoter sequence analysis of two interferon-activated humangenes <303> JOURNAL: Eur. J. Biochem. <304> VOLUME: 169 <305> ISSUE: 2<306> PAGES: 313-321 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Benech, P., et al. <302> TITLE:Interferon-responsive regulatory elements in the promoter of the human2′,5′-oligo(A) synthetase gene <303> JOURNAL: Mol. Cell. Biol. <304>VOLUME: 7 <305> ISSUE: 12 <306> PAGES: 4498-4504 <307> DATE: 1987 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Hovanessian,A.G., et al. <302> TITLE: Identification of 69-kd and 100-kd forms of2-5A sythetase <303> JOURNAL: EMBO J. <304> VOLUME: 6 <305> ISSUE: 5<306> PAGES: 1273-1280 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Williams, B.R., et al. <302> TITLE:Interferon-regulated human 2-5A synthetase gene maps to chromosome <303>JOURNAL: Somat. Cell Mol. Genet. <304> VOLUME: 12 <305> ISSUE: 4 <306>PAGES: 403-408 <307> DATE: 1986 <308> DATABASE ACCESSION NUMBER:NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Shiojiri, S., et al. <302> TITLE: Structureand expression of a cloned cDNA <303> JOURNAL: J. Biochem. <304> VOLUME:99 <305> ISSUE: 5 <306> PAGES: 1455-1464 <307> DATE: 1986 <308> DATABASEACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06<300> PUBLICATION INFORMATION: <301> AUTHORS: Wathelet, M., et al. <302>TITLE: Full-length sequence and expression of the 42 kDa 2-5A synthetase<303> JOURNAL: FEBS Lett. <304> VOLUME: 196 <305> ISSUE: 1 <306> PAGES:113-120 <307> DATE: 1986 <308> DATABASE ACCESSION NUMBER: NCBI/NM_016816<309> DATABASE ENTRY DATE: 2003-04-06 <300> PUBLICATION INFORMATION:<301> AUTHORS: Benech, P., et al. <302> TITLE: Structure of two forms ofthe interferon-induced (2′-5′) oligo A synthetase <303> JOURNAL: EMBO J.<304> VOLUME: 4 <305> ISSUE: 9 <306> PAGES: 2249-2256 <307> DATE: 1985<308> DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRYDATE: 2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Saunders,M.E., et al. <302> TITLE: Human 2-5A synthetase: characterization of anovel cDNA and corresponding gene structure <303> JOURNAL: EMBO J. <304>VOLUME: 4 <305> ISSUE: 7 <306> PAGES: 1761-1768 <307> DATE: 1985 <308>DATABASE ACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE:2003-04-06 <300> PUBLICATION INFORMATION: <301> AUTHORS: Merlin, G., etal. <302> TITLE: Molecular cloning and sequence of partial cDNA forinterferon-induced (2′-5′) oligo(A) synthetase mRNA from human cells<303> JOURNAL: Proc. Natl. Acad. Sci. U.S.A. <304> VOLUME: 80 <305>ISSUE: 16 <306> PAGES: 4904-4908 <307> DATE: 1983 <308> DATABASEACCESSION NUMBER: NCBI/NM_016816 <309> DATABASE ENTRY DATE: 2003-04-06<400> SEQUENCE: 13 gaggcagttc tgttgccact ctctctcctg tcaatgatggatctcagaaa taccccagcc 60 aaatctctgg acaagttcat tgaagactat ctcttgccagacacgtgttt ccgcatgcaa 120 atcgaccatg ccattgacat catctgtggg ttcctgaaggaaaggtgctt ccgaggtagc 180 tcctaccctg tgtgtgtgtc caaggtggta aagggtggctcctcaggcaa gggcaccacc 240 ctcagaggcc gatctgacgc tgacctggtt gtcttcctcagtcctctcac cacttttcag 300 gatcagttaa atcgccgggg agagttcatc caggaaattaggagacagct ggaagcctgt 360 caaagagaga gagcactttc cgtgaagttt gaggtccaggctccacgctg gggcaacccc 420 cgtgcgctca gcttcgtact gagttcgctc cagctcggggagggggtgga gttcgatgtg 480 ctgcctgcct ttgatgccct gggtcagttg actggcagctataaacctaa cccccaaatc 540 tatgtcaagc tcatcgagga gtgcaccgac ctgcagaaagagggcgagtt ctccacctgc 600 ttcacagaac tacagagaga cttcctgaag cagcgccccaccaagctcaa gagcctcatc 660 cgcctagtca agcactggta ccaaaattgt aagaagaagcttgggaagct gccacctcag 720 tatgccctgg agctcctgac ggtctatgct tgggagcgagggagcatgaa aacacatttc 780 aacacagccc aaggatttcg gacggtcttg gaattagtcataaactacca gcaactctgc 840 atctactgga caaagtatta tgactttaaa aaccccattattgaaaagta cctgagaagg 900 cagctcacga aacccaggcc tgtgatcctg gacccggcggaccctacagg aaacttgggt 960 ggtggagacc caaagggttg gaggcagctg gcacaagaggctgaggcctg gctgaattac 1020 ccatgcttta agaattggga tgggtcccca gtgagctcctggattctgct ggctgaaagc 1080 aacagtacag acgatgagac cgacgatccc aggacgtatcagaaatatgg ttacattgga 1140 acacatgagt accctcattt ctctcataga cccagcacgctccaggcagc atccacccca 1200 caggcagaag aggactggac ctgcaccatc ctctgaatgccagtgcatct tgggggaaag 1260 ggctccagtg ttatctggac cagttccttc attttcaggtgggactcttg atccagagaa 1320 gacaaagctc ctcagtgagc tggtgtataa tccaagacagaacccaagtc tcctgactcc 1380 tggccttcta tgccctctat cctatcatag ataacattctccacagcctc acttcattcc 1440 acctattctc tgaaaatatt ccctgagaga gaacagagagatttagataa gagaatgaaa 1500 ttccagcctt gactttcttc tgtgcacctg atgggagggtaatgtctaat gtattatcaa 1560 taacaataaa aataaagcaa ataccaaaaa 1590 <210>SEQ ID NO 14 <211> LENGTH: 3068 <212> TYPE: DNA <213> ORGANISM: Homosapiens <300> PUBLICATION INFORMATION: <301> AUTHORS: Hovnanian, A., etal. <302> TITLE: The human 2′,5′-oligoadenylate synthetase locus iscomposed of three distinct genes <303> JOURNAL: Genomics <304> VOLUME:52 <305> ISSUE: 3 <306> PAGES: 267-277 <307> DATE: 1998 <308> DATABASEACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03<300> PUBLICATION INFORMATION: <301> AUTHORS: Marie, I. and Hovanessian,A.G. <302> TITLE: The 69-kDa 2-5A synthetase is composed of twohomologous and adjacent functional domains <303> JOURNAL: J. Biol. Chem.<304> VOLUME: 267 <305> ISSUE: 14 <306> PAGES: 9933-9939 <307> DATE:1992 <308> DATABASE ACCESSION NUMBER: NCBI/NM_002535 <309> DATABASEENTRY DATE: 2003-04-03 <300> PUBLICATION INFORMATION: <301> AUTHORS:Marie, I., et al. <302> TITLE: Differential expression and distinctstructure of 69- and 100-kDa forms of 2-5A synthetase <303> JOURNAL: J.Biol. Chem. <304> VOLUME: 265 <305> ISSUE: 30 <306> PAGES: 18601-18607<307> DATE: 1990 <308> DATABASE ACCESSION NUMBER: NCBI/NM_002535 <309>DATABASE ENTRY DATE: 2003-04-03 <300> PUBLICATION INFORMATION: <301>AUTHORS: Marie, I., et al. <302> TITLE: Preparation and characterizationof polyclonal antibodies <303> JOURNAL: Biochem. Biophys. Res. Commun.<304> VOLUME: 160 <305> ISSUE: 2 <306> PAGES: 580-587 <307> DATE: 1989<308> DATABASE ACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRYDATE: 2003-04-03 <300> PUBLICATION INFORMATION: <301> AUTHORS:Hovanessian, A.G., et al. <302> TITLE: Characterization of 69- and100-kDa forms of 2-5A- synthetase <303> JOURNAL: J. Biol. Chem. <304>VOLUME: 263 <305> ISSUE: 10 <306> PAGES: 4959 <307> DATE: 1988 <308>DATABASE ACCESSION NUMBER: NCBI/NM_002535 <309> DATABASE ENTRY DATE:2003-04-03 <300> PUBLICATION INFORMATION: <301> AUTHORS: Hovanessian,A.G., et al. <302> TITLE: Identification of 69-kd and 100-kd forms of2-5A synthetase <303> JOURNAL: EMBO J. <304> VOLUME: 6 <305> ISSUE: 5<306> PAGES: 1273-1280 <307> DATE: 1987 <308> DATABASE ACCESSION NUMBER:NCBI/NM_002535 <309> DATABASE ENTRY DATE: 2003-04-03 <400> SEQUENCE: 14cggcagccag ctgagagcaa tgggaaatgg ggagtcccag ctgtcctcgg tgcctgctca 60gaagctgggt tggtttatcc aggaatacct gaagccctac gaagaatgtc agacactgat 120cgacgagatg gtgaacacca tctgtgacgt ctgcaggaac cccgaacagt tccccctggt 180gcagggagtg gccataggtg gctcctatgg acggaaaaca gtcttaagag gcaactccga 240tggtaccctt gtccttttct tcagtgactt aaaacaattc caggatcaga agagaagcca 300acgtgacatc ctcgataaaa ctggggataa gctgaagttc tgtctgttca cgaagtggtt 360gaaaaacaat ttcgagatcc agaagtccct tgatgggtcc accatccagg tgttcacaaa 420aaatcagaga atctctttcg aggtgctggc cgccttcaac gctctgagct taaatgataa 480tcccagcccc tggatctatc gagagctcaa aagatccttg gataagacaa atgccagtcc 540tggtgagttt gcagtctgct tcactgaact ccagcagaag ttttttgaca accgtcctgg 600aaaactaaag gatttgatcc tcttgataaa gcactggcat caacagtgcc agaaaaaaat 660caaggattta ccctcgctgt ctccgtatgc cctggagctg cttacggtgt atgcctggga 720acaggggtgc agaaaagaca actttgacat tgctgaaggc gtcagaacgg ttctggagct 780gatcaaatgc caggagaagc tgtgtatcta ttggatggtc aactacaact ttgaagatga 840gaccatcagg aacatcctgc tgcaccagct ccaatcagcg aggccagtaa tcttggatcc 900agttgaccca accaataatg tgagtggaga taaaatatgc tggcaatggc tgaaaaaaga 960agctcaaacc tggttgactt ctcccaacct ggataatgag ttacctgcac catcttggaa 1020tgtcctgcct gcaccactct tcacgacccc aggccacctt ctggataagt tcatcaagga 1080gtttctccag cccaacaaat gcttcctaga gcagattgac agtgctgtta acatcatccg 1140tacattcctt aaagaaaact gcttccgaca atcaacagcc aagatccaga ttgtccgggg 1200aggatcaacc gccaaaggca cagctctgaa gactggctct gatgccgatc tcgtcgtgtt 1260ccataactca cttaaaagct acacctccca aaaaaacgag cggcacaaaa tcgtcaagga 1320aatccatgaa cagctgaaag ccttttggag ggagaaggag gaggagcttg aagtcagctt 1380tgagcctccc aagtggaagg ctcccagggt gctgagcttc tctctgaaat ccaaagtcct 1440caacgaaagt gtcagctttg atgtgcttcc tgcctttaat gcactgggtc agctgagttc 1500tggctccaca cccagccccg aggtttatgc agggctcatt gatctgtata aatcctcgga 1560cctcccggga ggagagtttt ctacctgttt cacagtcctg cagcgaaact tcattcgctc 1620ccggcccacc aaactaaagg atttaattcg cctggtgaag cactggtaca aagagtgtga 1680aaggaaactg aagccaaagg ggtctttgcc cccaaagtat gccttggagc tgctcaccat 1740ctatgcctgg gagcagggga gtggagtgcc ggattttgac actgcagaag gtttccggac 1800agtcctggag ctggtcacac aatatcagca gctcggcatc ttctggaagg tcaattacaa 1860ctttgaagat gagaccgtga ggaagtttct actgagccag ttgcagaaaa ccaggcctgt 1920gatcttggac ccaggcgaac ccacaggtga cgtgggtgga ggggaccgtt ggtgttggca 1980tcttctggac aaagaagcaa aggttaggtt atcctctccc tgcttcaagg atgggactgg 2040aaacccaata ccaccttgga aagtgccggt aaaagtcatc taaaggaggc gttgtctgga 2100aatagccctg taacaggctt gaatcaaaga acttctccta ctgtagcaac ctgaaattaa 2160ctcagacaca aataaaggaa acccagctca caggagctta aacagctggt cagcccccct 2220aagcccccac tacaagtgat cctcaggcag gtaaccccag attcatgcac tgtagggctg 2280ggcgcagcat ccctaggtct ctacccagta gatgccacta gccctcctct cccagtgaca 2340accaaaagtc ttcacatgtt caaacgttcc cctgggttca cagatctttc tgcctttggc 2400ttttggctcc accctcttta gctgttaatt tgagtactta tggccctgaa agcggccacg 2460gtgcctccag atggcaggtt tgcaatccaa gcaggaagaa ggaaaagata cccaaaggtc 2520aagaacacag tgattttatt agaagtttca tccgcaaatt ttcttccatt tcattgctca 2580gaatgtcatg tggttacctg taacttgaag gtggctacaa agatgactgt ggaggtggtt 2640gcacttgcca cccaaggatg tctgccacac ctctccaagc cctcctacct accaagatat 2700acctgatata tccaccagat atctcctcag atatacttgg ttctctccac caggttcttt 2760ctttaaagca ggattctcaa ctttgatact tactcacatt gggctagaca gttctttgtt 2820tggaggctct cttgtgcatg taggatgttg agcagcatgt gtggcctgta cccagtacat 2880gccacccagt tgtgacaatt aaaagtgtct tgagacttta tcatgtgtct tctgccctag 2940gtgagaaccc ttgcactaca ggaaccctac acccaacctg gggggaatgt agggaagagg 3000tgccaagcca accgtggggt tagctctaat tattaagtta tgcattataa ataaatacca 3060aaaaattg 3068 <210> SEQ ID NO 15 <211> LENGTH: 6270 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <301>AUTHORS: Rebouillat, D., et al. <302> TITLE: The 100-kDa2′,5′-oligoadenylate synthetase catalyzing preferentially the synthesisof dimeric pppA2′p5′A molecules <303> JOURNAL: J. Biol. Chem. <304>VOLUME: 274 <305> ISSUE: 3 <306> PAGES: 1557-1565 <307> DATE: 1999 <308>DATABASE ACCESSION NUMBER: NCBI/AF063613 <309> DATABASE ENTRY DATE:1999-05-04 <300> PUBLICATION INFORMATION: <301> AUTHORS: Rebouillat, D.,and Hovanessian, A.G. <302> TITLE: Direct Submission <303> JOURNAL:Submitted (07-May-1998) Dept. of AIDS and Retroviruses, Institut Pasteur<304> VOLUME: 0 <305> ISSUE: 0 <306> PAGES: 0 <307> DATE: 1998 <308>DATABASE ACCESSION NUMBER: NCBI/AF063613 <309> DATABASE ENTRY DATE:1999-05-04 <400> SEQUENCE: 15 gccctgcttc cccttgcacc tgcgccgggcggccatggac ttgtacagca ccccggccgc 60 tgcgctggac aggttcgtgg ccagaaggctgcagccgcgg aaggagttcg tagagaaggc 120 gcggcgcgct ctgggcgccc tggccgctgccctgagggag cgcgggggcc gcctcggtgc 180 tgctgccccg cgggtgctga aaactgtcaagggaggctcc tcgggccggg gcacagctct 240 caagggtggc tgtgattctg aacttgtcatcttcctcgac tgcttcaaga gctatgtgga 300 ccagagggcc cgccgtgcag agatcctcagtgagatgcgg gcatcgctgg aatcctggtg 360 gcagaaccca gtccctggtc tgagactcacgtttcctgag cagagcgtgc ctggggccct 420 gcagttccgc ctgacatccg tagatcttgaggactggatg gatgttagcc tggtgcctgc 480 cttcaatgtc ctgggtcagg ccggctccgcggtcaaaccc aagccacaag tctactctac 540 cctcctcaac agtggctgcc aagggggcgagcatgcggcc tgcttcacag agctgcggag 600 gaactttgtg aacattcgcc cagccaagttgaagaaccta atcttgctgg tgaagcactg 660 gtaccaccag gtgtgcctac aggggttgtggaaggagacg ctgcccccgg tctatgccct 720 ggaattgctg accatcttcg cctgggagcagggctgtaag aaggatgctt tcagcctagg 780 cgaaggcctc cgaactgtcc tgggcctgatccaacagcat cagcacctgt gtgttttctg 840 gactgtcaac tatggcttcg aggaccctgcagttgggcag ttcttgcagc ggcacgttaa 900 gagacccagg cctgtgatcc tggacccagctgaccccaca tgggacctgg ggaatggggc 960 agcctggcac tgggatttgc atgcccaggaggcagcatcc tgctatgacc acccatgctt 1020 tctgaggggg atgggggacc cagtgcagtcttggaagggg ccgggccttc cacgtgctgg 1080 atgctcaggt ttgggccacc ccatccagctagaccctaac cagaagaccc ctgaaaacag 1140 caagagcctc aatgctgtgt acccaagagcagggagcaaa cctccctcat gcccagctcc 1200 tggccccact gcggagccag catcgtacccctctgtgccg ggaatggcct tggacctgtc 1260 tcagatcccc accaaggagc tggaccgcttcatccaggac cacctgaagc cgagccccca 1320 gttccaggag caggtgaaaa aggccatcgacatcatcttg cgctgcctcc atgagaactg 1380 tgttcacaag gcctcaagag tcagtaaagggggctcattt ggccggggca cagacctaag 1440 ggatggctgt gatgttgaac tcatcatcttcctcaactgc ttcacggact acaaggacca 1500 ggggccccgc cgcgcagaga tccttgatgagatgcgagcg cacgtagaat cctggtggca 1560 ggaccaggtg cccagcctga gccttcagtttcctgagcag aatgtgcctg aggctctgca 1620 gttccagctg gtgtccacag ccctgaagagctggacggat gttagcctgc tgcctgcctt 1680 cgatgctgtg gggcagctca gttctggcaccaaaccaaat ccccaggtct actcgaggct 1740 cctcaccagt ggctgccagg agggcgagcataaggcctgc ttcgcagagc tgcggaggaa 1800 cttcatgaac attcgccctg tcaagctgaagaacctgatt ctgctggtga agcactggta 1860 ccgccaggtt gcggctcaga acaaaggaaaaggaccagcc cctgcctctc tgcccccagc 1920 ctatgccctg gagctcctca ccatctttgcctgggagcag ggctgcaggc aggattgttt 1980 caacatggcc caaggcttcc ggacggtgctggggctcgtg caacagcatc agcagctctg 2040 tgtctactgg acggtcaact atagcactgaggacccagcc atgagaatgc accttcttgg 2100 ccagcttcga aaacccagac ccctggtcctggaccccgct gatcccacct ggaacgtggg 2160 ccacggtagc tgggagctgt tggcccaggaagcagcagcg ctggggatgc aggcctgctt 2220 tctgagtaga gacgggacat ctgtgcagccctgggatgtg atgccagccc tcctttacca 2280 aaccccagct ggggaccttg acaagttcatcagtgaattt ctccagccca accgccagtt 2340 cctggcccag gtgaacaagg ccgttgataccatctgttca tttttgaagg aaaactgctt 2400 ccggaattct cccatcaaag tgatcaaggtggtcaagggt ggctcttcag ccaaaggcac 2460 agctctgcga ggccgctcag atgccgacctcgtggtgttc ctcagctgct tcagccagtt 2520 cactgagcag ggcaacaagc gggccgagatcatctccgag atccgagccc agctggaggc 2580 atgtcaacag gagcggcagt tcgaggtcaagtttgaagtc tccaaatggg agaatccccg 2640 cgtgctgagc ttctcactga catcccagacgatgctggac cagagtgtgg actttgatgt 2700 gctgccagcc tttgacgccc taggccagctggtctctggc tccaggccca gctctcaagt 2760 ctacgtcgac ctcatccaca gctacagcaatgcgggcgag tactccacct gcttcacaga 2820 gctacaacgg gacttcatca tctctcgccctaccaagctg aagagcctga tccggctggt 2880 gaagcactgg taccagcagt gtaccaagatctccaagggg agaggctccc tacccccaca 2940 gcacgggctg gaactcctga ctgtgtatgcctgggagcag ggcgggaagg actcccagtt 3000 caacatggct gagggcttcc gcacggtcctggagctggtc acccagtacc gccagctctg 3060 tatctactgg accatcaact acaacgccaaggacaagact gttggagact tcctgaaaca 3120 gcagcttcag aagcccaggc ctatcatcctggatccggct gacccgacag gcaacctggg 3180 ccacaatgcc cgctgggacc tgctggccaaggaagctgca gcctgcacat ctgccctgtg 3240 ctgcatggga cggaatggca tccccatccagccatggcca gtgaaggctg ctgtgtgaag 3300 ttgagaaaat cagcggtcct actggatgaagagaagatgg acaccagccc tcagcatgag 3360 gaaattcagg gtcccctacc agatgagagagattgtgtac atgtgtgtgt gagcacatgt 3420 gtgcatgtgt gtgcacacgt gtgcatgtgtgtgttttagt gaatctgctc tcccagctca 3480 cacactcccc tgcctcccat ggcttacacactaggatcca gactccatgg tttgacacca 3540 gcctgcgttt gcagcttctc tgtcacttccatgactctat cctcatacca ccactgctgc 3600 ttcccaccca gctgagaatg ccccctcctccctgactcct ctctgcccat gcaaattagc 3660 tcacatcttt cctcctgctg caatccatcccttcctccca ttggcctctc cttgccaaat 3720 ctaaatactt tatataggga tggcagagagttcccatctc atctgtcagc cacagtcatt 3780 tggtactggc tacctggagc cttatcttctgaagggtttt aaagaatggc caattagctg 3840 agaagaatta tctaatcaat tagtgatgtctgccatggat gcagtagagg aaagtggtgg 3900 tacaagtgcc atgattgatt agcaatgtctgcactggata tggaaaaaag aaggtgcttg 3960 caggtttaca gtgtatatgt gggctattgaagagccctct gagctcggtt gctagcagga 4020 gagcatgccc atattggctt actttgtctgccacagacac agacagaggg agttgggaca 4080 tgcatgctat ggggaccctc ttgttggacacctaattgga tgcctcttca tgagaggcct 4140 ccttttcttc accttttatg ctgcactcctcccctagttt acacatcttg atgctgtggc 4200 tcagtttgcc ttcctgaatt tttattgggtccctgttttc tctcctaaca tgctgagatt 4260 ctgcatcccc acagcctaaa ctgagccagtggccaaacaa ccgtgctcag cctgtttctc 4320 tctgccctct agagcaaggc ccaccaggtccatccaggag gctctcctga cctcaagtcc 4380 aacaacagtg tccacactag tcaaggttcagcccagaaaa cagaaagcac tctaggaatc 4440 ttaggcagaa agggatttta tctaaatcactggaaaggct ggaggagcag aaggcagagg 4500 ccaccactgg actattggtt tcaatattagaccactgtag ccgaatcaga ggccagagag 4560 cagccactgc tactgctaat gccaccactacccctgccat cactgcccca catggacaaa 4620 actggagtcg agacctaggt tagattcctgcaaccacaaa catccatcag ggatggccag 4680 ctgccagagc tgcgggaaga cggatcccacctccctttct tagcagaatc taaattacag 4740 ccagacctct ggctgcagag gagtctgagacatgtatgat tgaatgggtg ccaagtgcca 4800 gggggcggag tccccagcag atgcatcctggccatctgtt gcgtggatga gggagtgggt 4860 ctatctcaga ggaaggaaca ggaaacaaagaaaggaagcc actgaacatc ccttctctgc 4920 tccacaggag tgtcttagac agcctgactctccacaaacc actgttaaaa cttacctgct 4980 aggaatgcta gattgaatgg gatgggaagagccttccctc attattgtca ttcttggaga 5040 gaggtgagca accaagggaa gctcctctgattcacctaga acctgttctc tgccgtcttt 5100 ggctcagcct acagagacta gagtaggtgaagggacagag gacagggctt ctaatacctg 5160 tgccatattg acagcctcca tccctgtcccccatcttggt gctgaaccaa cgctaagggc 5220 accttcttag actcacctca tcgatactgcctggtaatcc aaagctagaa ctctcaggac 5280 cccaaactcc acctcttgga ttggccctggctgctgccac acacatatcc aagagctcag 5340 ggccagttct ggtgggcagc agagacctgctctgccaagt tgtccagcag cagagtggcc 5400 ctggcctggg catcacaagc cagtgatgctcctgggaaga ccaggtggca ggtcgcagtt 5460 gggtaccttc cattcccacc acacagactctgggcctccc cgcaaaatgg ctccagaatt 5520 agagtaatta tgagatggtg ggaaccagagcaactcaggt gcatgataca aggagaggtt 5580 gtcatctggg tagggcagag aggagggcttgctcatctga acaggggtgt atttcattcc 5640 aggccctcag tctttggcaa tggccaccctggtgttggca tattggcccc actgtaactt 5700 ttgggggctt cccggtctag ccacaccctcggatggaaag acttgactgc ataaagatgt 5760 cagttctccc tgagttgatt gataggcttaatggtcaccc taaaaacacc cacatatgct 5820 tttcgatgga accagataag ttgacgctaaagttcttatg gaaaaataca cacgcaatag 5880 ctaggaaaac acagggaaag aagagttctgagcagggcct agtcttagcc aatattaaaa 5940 catactatga agcctctgat acttaaacagcatggcgctg gtacgtaaat agaccaatgc 6000 agttaggtgg ctctttccaa gactctggggaaaaaagtag taaaaagcta aatgcaatca 6060 atcagcaatt gaaagctaag tgagagagccagagggcctc cttggtggta aaagagggtt 6120 gcatttcttg cagccagaag gcagagaaagtgaagaccaa gtccagaact gaatcctaag 6180 aaatgcagga ctgcaaagaa attggtgtgtgtgtgtgtgt gtgtgtgtgt gtgtgtttaa 6240 tttttaaaaa gtttttattc ggaatccgcg6270 <210> SEQ ID NO 16 <211> LENGTH: 1412 <212> TYPE: DNA <213>ORGANISM: Mus musculus <300> PUBLICATION INFORMATION: <301> AUTHORS:Coccia, E.M., et al. <302> TITLE: A full-length murine 2-5A synthetasecDNA transfected in NIH-3T3 cells <303> JOURNAL: Virology <304> VOLUME:179 <305> ISSUE: 1 <306> PAGES: 228-233 <307> DATE: 1990 <308> DATABASEACCESSION NUMBER: NCBI/M33863 <309> DATABASE ENTRY DATE: 1993-06-11<400> SEQUENCE: 16 ccaggctggg agacccagga agctccagac ttagcatggagcacggactc aggagcatcc 60 cagcctggac gctggacaag ttcatagagg attacctccttcccgacacc acctttggtg 120 ctgatgtcaa atcagccgtc aatgtcgtgt gtgatttcctgaaggagaga tgcttccaag 180 gtgctgccca cccagtgagg gtctccaagg tggtgaagggtggctcctca ggcaaaggca 240 ccacactcaa gggcaggtca gacgctgacc tggtggtgttccttaacaat ctcaccagct 300 ttgaggatca gttaaaccga cggggagagt tcatcaaggaaattaagaaa cagctgtacg 360 aggttcagca tgagagacgt tttagagtca agtttgaggtccagagttca tggtggccca 420 acgcccggtc tctgagcttc aagctgagcg ccccccatctgcatcaggag gtggagtttg 480 atgtgctgcc agcctttgat gtcctgggtc atgttaatacttccagcaag cctgatccca 540 gaatctatgc catcctcatc gaggaatgta cctccctggggaaggatggc gagttctcta 600 cctgcttcac ggagctccag cggaacttcc tgaagcagcgcccaaccaag ctgaagagtc 660 tcatccgcct ggtcaagcac tggtaccaac tgtgtaaggagaagctgggg aagccattgc 720 ctccacagta cgccctagag ttgctcactg tctttgcctgggaacaaggg aatggatgtt 780 atgagttcaa cacagcccag ggcttccgga ccgtcttggaactggtcatc aattatcagc 840 atcttcgaat ctactggaca aagtattatg actttcaacaccaggaggtc tccaaatacc 900 tgcacagaca gctcagaaaa gccaggcctg tgatcctggacccagctgac ccaacaggga 960 atgtggccgg tgggaaccca gagggctgga ggcggttggctgaagaggct gatgtgtggc 1020 tatggtaccc atgttttatt aaaaaggatg gttcccgagtgagctcctgg gatgtgccga 1080 cggtggttcc tgtacctttt gagcaggtag aagagaactggacatgtatc ctgctgtgag 1140 cacagcagca cctgcccagg agactgctgg tcaggggcatttgctgctct gctgcaggcc 1200 catgacccag tgagggaggg ccccacctgg catcagactccgtgcttctg atgcctgcca 1260 gccatgtttg actcctgtcc aatcacagcc agccttcctcaacagattca gaaggagagg 1320 aaagaacaca cgcttggtgt ccatctgtcc acctgttggaaggttctgtc tgacaaagtc 1380 tgatcaacaa taaaccacag caggtgccgt ca 1412<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseODN (p40 subunit) <400> SEQUENCE: 17 tttctgagat ccatcattga 20 <210> SEQID NO 18 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Antisense ODN (p69subunit) <400> SEQUENCE: 18 tccccatttc ccattgc 17 <210> SEQ ID NO 19<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Control ODN sequence <400>SEQUENCE: 19 gtctatgaat actttcctag 20 <210> SEQ ID NO 20 <211> LENGTH:17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Control ODN sequence <400> SEQUENCE: 20cacctctatc tctctcg 17

What is claimed is:
 1. A method of inhibiting an RNA virus infection ina patient by increasing the endogenous 2′-5′ oligoadenylate synthetaseactivity within the patient, wherein the RNA virus is a type thattransiently produces double-stranded RNA during intermediatereplication.
 2. The method according to claim 1, wherein said increasingcomprises administering a nucleotide sequence encoding a 2′-5′oligoadenylate synthetase, or a catalytically active fragment thereof,to the patient, wherein the nucleotide sequence is expressed in thepatient.
 3. The method according to claim 1, wherein said increasingcomprises administering a nucleotide sequence encoding at least onecatalytically active fragment of 2′-5′ oligoadenylate synthetase to thepatient, wherein the catalytically active fragment comprises acatalytically active subunit of 2′-5′ oligoadenylate synthetase selectedfrom the group consisting of p40, p69, and plOO, wherein the nucleotidesequence is expressed in the patient.
 4. The method according to claim2, wherein the 2′-5′ oligoadenylate synthetase is a human enzyme ormammalian homologue.
 5. The method according to claim 2, wherein thenucleotide sequence encodes a polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, AND SEQ ID NO: 12, or a catalytically active fragment of anyof the foregoing.
 6. The method according to claim 2, wherein thenucleotide sequence comprises at least one sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or a catalytically activefragment of any of the foregoing.
 7. The method according to claim 2,wherein the nucleotide sequence comprises SEQ ID NO. 1, or acatalytically active fragment thereof.
 8. The method according to claim2, wherein the nucleotide sequence comprises SEQ ID NO. 3, or acatalytically active fragment thereof.
 9. The method according to claim2, wherein the nucleotide sequence comprises SEQ ID NO. 5, or acatalytically active fragment thereof.
 10. The method according to claim2, wherein the nucleotide sequence comprises SEQ ID NO. 7, or acatalytically active fragment thereof.
 11. The method according to claim1, wherein the RNA virus is a member of the family paramyxoviridae. 12.The method according to claim 1, wherein the RNA virus is selected fromthe group consisting of respiratory syncytial virus, rhinovirus,vaccinia virus, reovirus, HIV, EMCV, hepatitis B, hepatitis C, bovinerespiratory syncytial virus, measles virus, sendai virus, parainfluenzavirus, mumps virus, simian virus, newcastle virus, coronavirus, and WestNile virus.
 13. The method according to claim 1, wherein the RNA virusis coronavirus or West Nile virus.
 14. The method according to claim 1,wherein the RNA virus is one in which exposure to interferon activelyinhibits viral replication.
 15. The method according to claim 1, whereinthe RNA virus is respiratory syncytial virus.
 16. The method accordingto claim 1, wherein the patient is human.
 17. The method according toclaim 1, wherein the patient is a non-human animal.
 18. The methodaccording to claim 1, wherein the patient is suffering from the RNAvirus infection, and wherein the nucleotide sequence alleviates at leastone of the symptoms associated with the RNA virus infection.
 19. Themethod according to claim 1, wherein the patient is not suffering fromthe RNA virus infection.
 20. The method according to claim 2, whereinthe nucleotide sequence is expressed within the patient, therebyeliciting a physiological response from the patient selected from thegroup consisting of: reduction of respiratory syncytial viral titerswithin the patient's lungs; reduction of MIP 1-a chemokine, decrease inbronchioalveolar lavage lymphocytes and macrophages, reduction inepithelial cell damage, reduction in infiltration of mononuclear cellsin the peribronchiolar and perivascular regions, and reduction inthickness of the patient's alveolar septa.
 21. The method according toclaim 2, wherein the nucleotide sequence is administered to the patientwithin a vector, wherein the vector comprises the nucleotide sequenceoperably linked to a promoter sequence, and wherein the promotersequence drives expression of the nucleotide sequence.
 22. The methodaccording to claim 21, wherein the vector is a viral vector.
 23. Themethod according to claim 22, wherein the viral vector is adenovirus oradeno-associated virus.
 24. The method according to claim 21, whereinthe vector is a non-viral vector.
 25. The method according to claim 21,wherein the vector is a plasmid.
 26. The method according to claim 2,wherein the nucleotide sequence is administered to the patient orally orintranasally.
 27. The method according to claim 2, wherein thenucleotide sequence is administered with a pharmaceutically acceptablecarrier.
 28. The method according to claim 27, wherein thepharmaceutically acceptable carrier comprises chitosan or a derivativethereof.
 29. The method according to claim 1, wherein said increasingcomprises administering a 2′-5′ oligoadenylate synthetase, or at leastone catalytically active fragment thereof, to the patient.
 30. Themethod according to claim 1, wherein said increasing comprisesadministering at least one catalytically active fragment of 2′-5′oligoadenylate synthetase to the patient, wherein the catalyticallyactive fragment comprises a catalytically active subunit of 2′-5′oligoadenylate synthetase selected from the group consisting of p40,p69, and p100.
 31. The method according to claim 29, wherein the 2′-5′oligoadenylate synthetase is a human enzyme or mammalian homologue. 32.The method according to claim 29, wherein a catalytically activefragment of 2′-5′ oligoadenylate synthetase is administered to thepatient, wherein the catalytically active fragment comprises at leastone amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ IDNO: 12, or a catalytically active fragment of any of the foregoing. 33.The method according to claim 32, wherein the amino acid sequenceadministered to the patient comprises SEQ ID NO: 2, or a catalyticallyactive fragment thereof.
 34. The method according to claim 32, whereinthe amino acid sequence administered to the patient comprises SEQ ID NO:4, or a catalytically active fragment thereof.
 35. The method accordingto claim 32, wherein the amino acid sequence administered to the patientcomprises SEQ ID NO: 6, or a catalytically active fragment thereof. 36.The method according to claim 32, wherein the amino acid sequenceadministered to the patient comprises SEQ ID NO: 8, or a catalyticallyactive fragment thereof.
 37. The method according to claim 29, whereinthe 2′-5′ oligoadenylate synthetase, or at least one catalyticallyactive fragment thereof, is administered to the patient with apharmaceutically acceptable carrier.
 38. The method according to claim29, wherein the 2′-5′ oligoadenylate synthetase, or at least onecatalytically active fragment thereof, is administered to the patientwithin a composition that protects the 2′-5′ oligoadenylate synthetase,or at least one catalytically active fragment thereof, from prematureproteolytic degradation within the patient.
 39. A pharmaceuticalcomposition comprising a nucleotide sequence encoding a 2′-5′oligoadenylate synthetase, or at least one catalytically active fragmentthereof, and a pharmaceutically acceptable carrier.
 40. The compositionof claim 39, wherein said composition further comprises an anti-viralagent.
 41. The pharmaceutical composition of claim 39, wherein saidnucleotide sequence encodes a polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, AND SEQ ID NO: 12, or a catalytically active fragment of anyof the foregoing.
 42. The pharmaceutical composition of claim 39,wherein the nucleotide sequence comprises at least one sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16, or a catalytically active fragment of any ofthe foregoing.
 43. A pharmaceutical composition comprising a 2′-5′oligoadenylate synthetase, or at least one catalytically active fragmentthereof, and a pharmaceutically acceptable carrier.
 44. Thepharmaceutical composition of claim 43, wherein the compositioncomprises a polypeptide selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ IDNO:12, or a catalytically active fragment of the foregoing.
 45. A vectorcomprising a nucleotide sequence encoding a 2′-5′ oligoadenylatesynthetase, or at least one catalytically active fragment thereof, and apromoter sequence operably linked to said nucleotide sequence.
 46. Thevector of claim 46, wherein said nucleotide sequence encodes apolypeptide selected from the group consisting of SEQ ID NO: 2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, AND SEQ ID NO: 12, or acatalytically active fragment of any of the foregoing.
 47. The vector ofclaim 45, wherein said nucleotide sequence comprises at least onesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, and SEQ ID NO:16, or a catalytically activefragment of any of the foregoing.
 48. The vector of claim 45, whereinsaid vector is a viral vector.
 47. The vector of claim 45, wherein saidviral vector is an adenoviral vector or adeno-associated viral vector.48. A host cell that has been genetically modified with a nucleotidesequence encoding a 2′-5′ oligoadenylate synthetase, or at least onecatalytically active fragment thereof, wherein said nucleotide sequenceis expressed in said cell.
 49. The host cell of claim 48, wherein saidnucleotide sequence encodes a polypeptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:lO, AND SEQ ID NO: 12, or a catalytically active fragment of anyof the foregoing.
 50. The host cell of claim 48, wherein said nucleotidesequence comprises at least one sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ IDNO: 16, or a catalytically active fragment of any of the foregoing. 51.The host cell of claim 48, wherein said host cell is a prokaryotic cell.52. The host cell of claim 48, wherein said host cell is a eukaryoticcell.
 53. The host cell of claim 48, wherein said host cell is amammalian cell.
 54. The host cell of claim 48, wherein said host cell isa human cell.